Hybrid cyclic libraries and screens thereof

ABSTRACT

Provided are novel types of hybrid cyclic libraries that contain a known protein binding domain of a natural product. Also provided are synthetic methods to make such libraries and methods for the deconvolution of hits using partially split-pooled library compounds. Such methods are applicable for use with the entire human proteome to screen such libraries that bind and for the identification of hits.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 15/728,282 filed Oct. 9, 2017, now issued as U.S. Pat. No.10,414,801; which is a continuation application of U.S. application Ser.No. 14/987,653 filed Jan. 4, 2016, now issued as U.S. Pat. No.9,783,577; which is a continuation application of U.S. application Ser.No. 13/990,396 filed Jul. 1, 2013, now issued as U.S. Pat. No.9,250,237; which is a 35 USC § 371 National Stage application ofInternational Application No. PCT/US2011/062471 filed Nov. 29, 2011, nowexpired; which claims the benefit under 35 USC § 119(e) to U.S.Application Ser. No. 61/418,038 filed Nov. 30, 2010, now expired. Thedisclosure of each of the prior applications is considered part of andis incorporated by reference in the disclosure of this application.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No. CA174428awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION Field of the Invention

The invention relates generally to hybrid cyclic molecules, and morespecifically to hybrid cyclic libraries based on the immunophilin ligandfamily of natural products cyclosporine A (CsA), FK506, and rapamycyin,and methods of screening proteins encoded by a genome on a protein chipor in cell- and target-based assays for elucidation of the proteins'function.

Background Information

The immunophilin ligand family consists of three members, cyclosporine A(CsA), FK506 and rapamycin, all of which are natural products withpotent immunosuppressive or anticancer activities. Unlike otherbioactive small molecules, these natural products have an unprecedentedand extraordinary mode of action through induction of dimeric ternarycomplexes between two distinct proteins. They each bind to abundant andsmall cytosolic immunophilins, which also possess peptidyl prolylcis-trans isomerase activity and are implicated in protein folding.Thus, CsA binds the cyclophilin (CyP) family of immunophilins; FK506 andrapamcyin both bind FKBP. The formation of the immunophilin-drugcomplexes per se does not have significant cellular consequences. It isthe subsequent binding of these complexes to their respective targetproteins that leads to inhibition of T cell activation or tumor cellgrowth. In the case of CsA and FK506, the CyP-CsA and FKBP-FK506complexes bind to and inhibit the enzymatic activity of the proteinphosphatase calcineurin. In the case of rapamycin, the FKBP-rapamycincomplex binds to the PI3 kinase homologue, Target of Rapamycin (TOR).There are a number unique properties associated with this family ofnatural products. First, they are capable of targeting a relativelylarge surface of target protein through recruitment of the correspondingimmunophilins, capable of inhibiting protein-protein interactions inaddition to enzymatic activity of individual protein targets. Second,through their association with immunophilins, they are more stable andless susceptible to degradation in vivo through interaction withimmunophilins in both blood and in red blood cells. Third, theimmunophilin-binding domains confer intrinsic stabilities to themacrocycles. Thus, macrocyles containing the FKBP- or CyP-bindingdomains have great potential as new leads for developing drugs to beused for treating diseases.

With the completion of the sequencing and annotation of the humangenome, we now have a complete catalog of all human proteins encoded inthe genome. The functions of a majority of these proteins, however,remain unknown. One way to elucidate the functions of these proteins isto find small molecule ligands that specifically bind to the proteins ofinterest and perturb their biochemical and cellular functions. Thus, amajor challenge for chemical biologists today is to discover new smallmolecule probes for new proteins to facilitate the elucidation of theirfunctions. The recent advance in the development of protein chips hasoffered an exciting new opportunity to simultaneously screen chemicallibraries against nearly the entire human proteome. A single chip, inthe form of a glass slide, is sufficient to display an entire proteomein duplicate arrays. Recently, a protein chip with 17,000 human proteinsdisplayed on a single slide has been produced. A major advantage ofusing human protein chips for screening is that the entire displayedproteome can be interrogated at once in a small volume of assay buffer(<3 mL). Screening of human protein chips, however, is not yet feasiblewith most, if not all, existing chemical libraries due to the lack of auniversal readout for detecting the binding of a ligand to a protein onthese chips. While it is possible to add artificial tags to individualcompounds in a synthetic library, often the added tags themselvesinterfere with the activity of ligands. Thus, there remains a need fornew compounds and methods for screening chemical libraries against thehuman proteome.

SUMMARY OF THE INVENTION

The present invention solves these problems and others by providing newcompounds and methods for screening chemical libraries against theproteins encoded by a genome.

Thus, in one embodiment, the disclosure provides a compound of FormulaI:

or a pharmaceutically acceptable salt or solvate thereof, wherein:

is a single or double bond;

X₁ is O or NR⁶;

Y is —C(O)— or

X₂ is (CH₂)_(m), O, or NR⁶;

Z is

W is O, CH, CH₂, CR⁴, or CR⁵;

L₁ and L₂ are each independently a direct bond, substituted orunsubstituted —(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)O(C₁-C₆)alkyl-, substituted or unsubstituted —(CH₂)_(n)C(O)—,substituted or unsubstituted —(CH₂)_(n)C(O)(C₁-C₆)alkyl-, substituted orunsubstituted —(CH₂)_(n)C(O)O(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)NH(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)S(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₁-C₆)alkyl-, substituted or unsubstituted—(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)O(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)C(O)O(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)NH(C₁-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)S(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkenyl-, substituted or unsubstituted—(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)O(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)C(O)O(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)NH(C₁-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)S(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkynyl-, wherein each alkyl, alkenyl andalkynyl group may be optionally substituted with alkyl, alkoxy, amino,carboxyl, cyano, nitro, or trifluoromethyl;

each m is independently an integer selected from 0, 1, 2, 3, 4, 5, and6;

each n is independently an integer selected from 0, 1, 2, 3, 4, 5, and6;

R¹ is hydrogen, hydroxyl, or OPG, wherein PG is a protecting group, or

R¹ is

wherein

is a resin;

R² is hydrogen, hydroxyl, or alkoxy;

R³ is hydrogen or alkyl;

R⁴ and R⁵ are each independently hydrogen, hydroxy, alkyl, alkoxy, orOPG, wherein PG is a protecting group;

R⁶ is hydrogen or alkyl;

wherein the Effector Domain has Formula II:

wherein:

R⁷, R⁹, R¹¹, R¹³, and R¹⁵ are each independently hydrogen or alkyl;

R⁸, R¹⁰, R¹², and R¹⁴ are each independently hydrogen, halogen, amino,cyano, nitro, trifluoromethyl, substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted perfluoroalkyl, substituted or unsubstituted alkoxy,substituted or unsubstituted alkylamino, substituted or unsubstitutedalkylthio, substituted or unsubstituted aryl, substituted orunsubstituted alkylaryl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted heteroaryl, substituted or unsubstituted heteroalkylaryl,(CH₂)_(n)CN, (CH₂)_(n)CF₃, (CH₂)_(n)C₂F₅, (CH₂)_(n)OR¹⁶,(CH₂)_(n)C(O)R¹⁶, (CH₂)_(n)C(O)OR¹⁶, (CH₂)_(n)OC(O)R¹⁶,(CH₂)_(n)NR¹⁷R¹⁸, (CH₂)_(n)C(O)NR¹⁷R¹⁸, (CH₂)_(n)N¹⁹RC(O)R¹⁶,(CH₂)_(n)N¹⁹RC(O)OR¹⁶, (CH₂)_(n)NR¹⁹C(O)NR¹⁷R¹⁸, (CH₂)_(n)SR¹⁶,(CH₂)_(n)S(O)₃NR¹⁷R¹⁸, (CH₂)_(n)N¹⁹RS(O)_(j)R¹⁶, or—(CH₂)_(n)NR¹⁹S(O)_(j)NR¹⁷R¹⁸;

n is an integer selected from 0, 1, 2, 3, 4, 5, and 6;

j is an integer selected from 0, 1, and 2;

R¹⁶, R¹⁷, R¹⁸, and R¹⁹ are each independently hydrogen, halogen, amino,cyano, nitro, trifluoromethyl, alkyl, alkenyl, alkynyl, cycloalkyl,perfluoroalkyl, alkoxy, alkylamino, alkylthio, aryl, alkylaryl,heteroalkyl, heterocycloalkyl, heteroaryl, or heteroalkylaryl, or

R¹⁶ and R¹⁹ are as described above, and R¹⁷ and R¹⁸, together with the Natom to which they are attached, form a substituted or unsubstituted 5-,6-, or 7-membered heterocycloalkyl or a substituted or unsubstituted5-membered heteroaryl,

wherein each of the above groups listed for R⁸, R¹⁰, R¹², and R¹⁴ may beoptionally independently substituted with 1 to 3 groups selected fromhalogen, amino, cyano, nitro, trifluoromethyl, alkyl, alkenyl, alkynyl,cycloalkyl, perfluoroalkyl, alkoxy, alkylamino, alkylthio, aryl,alkylaryl, heteroalkyl, heterocycloalkyl, heteroaryl, heteroalkylaryl,(CH₂)_(n)CN, (CH₂)_(n)CF₃, (CH₂)_(n)C₂F₅, (CH₂)_(n)OR¹⁶,(CH₂)_(n)C(O)R¹⁶, (CH₂)_(n)C(O)OR¹⁶, (CH₂)_(n)OC(O)R¹⁶,(CH₂)_(n)NR¹⁷R¹⁸, (CH₂)_(n)C(O)NR¹⁷R¹⁸, (CH₂)_(n)N¹⁹RC(O)R¹⁶,(CH₂)_(n)N¹⁹RC(O)OR¹⁶, (CH₂)_(n)NR¹⁹C(O)NR¹⁷R¹⁸, (CH₂)_(n)SR¹⁶,(CH₂)_(n)S(O)_(j)NR¹⁷R¹⁸, (CH₂)_(n)N¹⁹RS(O)_(j)R¹⁶, and—(CH₂)_(n)NR¹⁹S(O)_(j)NR¹⁷R¹⁸.

In another embodiment, the disclosure provides methods for treatingcancer in a patient in need thereof by administering a compound ofFormula I to the patient.

In another embodiment, the disclosure provides methods for suppressingthe immune system in a patient in need thereof by administering thecompound of Formula I to the patient.

In another embodiment, the disclosure provides methods for preparing thecompound of Formula I.

In another embodiment, the disclosure provides methods for determiningthe function of a protein encoded in the human genome by:

a) screening a hybrid combinatorial peptide or non-peptide library ofcompounds that includes the FKBP-binding domain (FKBP) of the naturalproduct rapamycin or FK506 against the proteins encoded in the humangenome using a human protein chip;

b) detecting the binding of a compound to a protein on the chip usingthe anti-V5 antibody together with a fluorescently tagged secondaryantibody;

c) recording the fluorescence pattern of the human protein chip on achip reader;

d) identifying the proteins based on the physical location of thefluorescent spots on the chip; and

e) determining the function of the protein based on its perturbedbiochemical and cellular functions.

In another embodiment, the disclosure provides methods for generating alead compound as a high-affinity ligand of the FKBP isoforms, the methodcomprising the steps of:

a) screening a hybrid combinatorial peptide or non-peptide library ofcompounds that includes the FKBP-binding domain (FKBP) of the naturalproduct rapamycin or FK506 against the proteins encoded in the humangenome using a human protein chip;

b) detecting the binding of a compound to a protein on the chip usingthe anti-V5 antibody together with a fluorescently tagged secondaryantibody;

c) recording the fluorescence pattern of the human protein chip on achip reader;

d) identifying the proteins based on the physical location of thefluorescent spots on the chip; and

e) determining the structure of the lead compound.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows rapamycin and FK506 and their respective cellular targets.

FIG. 2 shows the two orthogonally split and pooled libraries.

FIG. 3 shows the detection of hits on protein chips.

DETAILED DESCRIPTION OF THE INVENTION

Abbreviations used herein have their conventional meaning within thechemical and biological arts.

As used herein, the terms “a,” “an,” or “a(n),” when used in referenceto a group of substituents, mean at least one. For example, where acompound is substituted with “an” alkyl or “an” aryl, the compound isoptionally substituted with at least one alkyl and/or at least one arylgroup. Moreover, where a moiety is substituted with a R substituent, thegroup may be referred to as “R-substituted.” Where a moiety isR-substituted, the moiety is substituted with at least one R substituentand each R substituent is optionally different.

The description of compounds of the present invention are limited byprinciples of chemical bonding known to those skilled in the art.Accordingly, where a group may be substituted by one or more of a numberof substituents, such substitutions are selected so as to comply withprinciples of chemical bonding and to give compounds which are notinherently unstable and/or would be known to one of ordinary skill inthe art as likely to be unstable under ambient conditions, such asaqueous, neutral, and several known physiological conditions. Forexample, a heterocycloalkyl or heteroaryl is attached to the remainderof the molecule via a ring heteroatom in compliance with principles ofchemical bonding known to those skilled in the art thereby avoidinginherently unstable compounds.

The symbol “—” and “˜”denote the point of attachment of a moiety to theremainder of the molecule.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left, e.g., —CH₂O— is equivalent to —OCH₂—.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight (i.e., unbranched) or branchedchain, or cyclic hydrocarbon radical, or combination thereof, which maybe fully saturated, mono- or polyunsaturated and can include di- andmultivalent radicals, having the number of carbon atoms designated(i.e., C₁-C₆ means one to six carbons; and C₁-C₁₀ means one to tencarbons).

Examples of saturated hydrocarbon radicals include, but are not limitedto, groups such as methyl, ethyl, N-propyl, isopropyl, N-butyl, t-butyl,isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl,homologs and isomers of, for example, N-pentyl, N-hexyl, N-heptyl,N-octyl, and the like. An unsaturated alkyl group is one having one ormore double bonds or triple bonds for example, alkenyl and alkynylgroups, respectively.

Examples of unsaturated alkyl groups include, but are not limited to,vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkyl, as exemplified, but not limited,by —CH═CH₂—, —CH₂CH═CH₂—, —CH₂CH═CHCH₂—, —CH₂CH₂CH═CH₂—, and—CH═CHCH(CH₂CH₂CH₃)CH₂—. Typically, an alkyl (or alkylene) group willhave from 1 to 24 carbon atoms, while other alkyl (or alkylene) groupswill have 10 or fewer carbon atoms. A “lower alkyl” or “lower alkylene”is a shorter chain alkyl or alkylene group, generally having eight orfewer carbon atoms, typically one to six carbon atoms.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of atleast one carbon atoms and at least one heteroatom selected from thegroup consisting of O, N, P, Si and S, and wherein the nitrogen,phosphorus, and sulfur atoms may optionally be oxidized and the nitrogenheteroatom may optionally be quaternized. The heteroatom(s) O, N, P andS and Si may be placed at any interior position of the heteroalkyl groupor at the position at which alkyl group is attached to the remainder ofthe molecule.

Examples of heteroalkyl include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂— S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,—CH═CH—N(CH₃)—CH₃, O—CH₃, —O—CH₂— CH₃ and —CN. In addition, up to two orthree heteroatoms may be consecutive, such as, for example, —CH₂—NH—OCH₃and —CH₂—O—Si(CH₃)₃.

The term “heteroalkylene” by itself or as part of another substituentmeans a divalent radical derived from heteroalkyl, as exemplified, butnot limited by, —CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. Forheteroalkylene groups, heteroatoms can also occupy either or both of thechain termini (e.g., alkyleneoxo, alkylenedioxo, alkyleneamino,alkylenediamino, and the like). Still further, for alkylene andheteroalkylene linking groups, no orientation of the linking group isimplied by the direction in which the formula of the linking group iswritten. For example, the formula —C(O)OR′— represents both —C(O)OR′—and —R′OC(O)—. As described above, heteroalkyl groups, as used herein,include those groups that are attached to the remainder of the moleculethrough a heteroatom, such as —C(O)R′, —C(O)NR′, —NR′R″, —OR′, —SR′,and/or —SO₂R′. Where “heteroalkyl” is recited, followed by recitationsof specific heteroalkyl groups, such as —NR′R″ and the like, it isunderstood that the terms heteroalkyl and —NR′R″ are not redundant ormutually exclusive. Rather, the specific heteroalkyl groups are recitedto add clarity. Thus, the term “heteroalkyl” should not be interpretedherein as excluding specific heteroalkyl groups, such as —NR′R″ and thelike.

The terms “cycloalkyl” and “heterocycloalkyl,” by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl,” respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like. The terms “cycloalkylene”and “heterocycloalkylene” refer to the divalent derivatives ofcycloalkyl and heterocycloalkyl, respectively.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” is mean to include, but not be limited to,trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, andthe like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent which can be a single ring or multiplerings (usually from 1 to 3 rings) which are fused together or linkedcovalently. The term “heteroaryl” refers to aryl groups (or rings) thatcontain from one to four heteroatoms (in each separate ring in the caseof multiple rings) selected from N, O, and S, wherein the nitrogen andsulfur atoms are optionally oxidized, and the nitrogen atom(s) areoptionally quaternized.

A heteroaryl group can be attached to the remainder of the moleculethrough a carbon or heteroatom. Non-limiting examples of aryl andheteroaryl groups include but are not limited to phenyl, 1-naphthyl,2-naphthyl, biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of above noted aryland heteroaryl ring systems are selected from the group of acceptablesubstituents described below. The terms “arylene” and “heteroarylene”refer to the divalent radicals of aryl and heteroaryl, respectively.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxo, arylthioxo, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, naphthyloxy)propyl, and the like).

Where a heteroalkyl, heterocycloalkyl, or heteroaryl includes a specificnumber of members (e.g., “3 to 7 membered”), the term “member” referrersto a carbon or heteroatom.

The term “oxo” as used herein means an oxygen that is double bonded to acarbon atom.

Each of above terms (e.g., “alkyl,” “heteroalkyl,” “cycloalkyl, and“heterocycloalkyl,” “aryl,” “heteroaryl” as well as their divalentradical derivatives) are meant to include both substituted andunsubstituted forms of the indicated radical. Examples of substituentsfor each type of radical are provided below.

Substituents for alkyl, heteroalkyl, cycloalkyl, heterocycloalkylmonovalent and divalent derivative radicals (including those groupsoften referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl,alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —C(O)NR′R″,—OC(O)NR′R″, —NR′C(O)R″, —NR′—C(O)NR″R′″, —NR′C(O)OR″,—NR′—C(NR″R″′)═NR″″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NR′SO₂R″, —CN and—NO₂ in a number ranging from zero to (2m′+1), where m¹ is the totalnumber of carbon atoms in such radical. R′, R″, R″′ and R″ eachindependently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g.,aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl,alkoxy or thioalkoxy groups, or arylalkyl groups. When a compound of theinvention includes more than one R group, for example, each of the Rgroups is independently selected as are each R′, R″, R′″ and R″″ groupswhen more than one of these groups is present. When R′ and R″ areattached to the same nitrogen atom, they can be combined with thenitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example,—NR′R″ is meant to include, but not be limited to, 1-pyrrolidinyl and4-morpholinyl. From the above discussion of substituents, one of skillin the art will understand that the term “alkyl” is meant to includegroups including carbon atoms bound to groups other than hydrogengroups, such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g.,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similar to the substituents described for alkyl radicals above,exemplary substituents for aryl and heteroaryl groups (as well as theirdivalent derivatives) are varied and are selected from, for example:halogen, —OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R, —C(O)R′,—CO₂R, —C(O)NR′R″, —OC(O)NR′R″, —NR¹C(O)R″, —NR′—C(O)NR″R″′,—NR′C(O)OR″, —NR′—C(NR′N″R″′)═NR″″, —NR′—C(NR′R″)═NR′″, —S(O)R′,—S(O)₂R′, —S(O)₂NR′R″, —NR′SO₂R″, —CN and —NO₂, —N₃, —CH(Ph)₂,fluoro(C₁-C₄)alkoxy, in a number ranging from zero to the total numberof open valences on aromatic ring system; and where R′, R″, R″′ and R″″are each independently selected from hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl and substituted orunsubstituted heteroaryl. When a compound of the invention includes morethan one R group, for example, each of the R groups is independentlyselected as are each R′, R″, R″′ and R″″ groups when more than one ofthese groups is present.

Two of the substituents on adjacent atoms of aryl or heteroaryl ring mayoptionally form a ring of the formula -T-C(O)—(CR′R″)_(q)—U—, wherein Tand U are independently —NR—, —O—, —CR′R″— or a single bond, and q is aninteger of from 0 to 3. Alternatively, two of the substituents onadjacent atoms of aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula -A-(CH₂)_(r)—B—, wherein A and B areindependently —CR′R″—, —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or asingle bond, and r is an integer of from 1 to 4. One of the single bondsof the new ring so formed may optionally be replaced with a double bond.Alternatively, two of the substituents on adjacent atoms of aryl orheteroaryl ring may optionally be replaced with a substituent of theformula —(CR′R″)_(s)—X¹—(CR″′R″″)_(d)—, where s and d are independentlyintegers of from 0 to 3, and X¹ is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or—S(O)₂NR′—. The substituents R′, R″, R″′ and R″″ are independentlyselected from hydrogen, substituted or unsubstituted alkyl, substitutedor unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl.

As used herein, the term “heteroatom” or “ring heteroatom” is meant toinclude oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), andsilicon (Si).

An “aminoalkyl” as used herein refers to an amino group covalently boundto an alkylene linker. The amino group is —NR′R″, wherein R′ and R″ aretypically selected from hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl.

A “substituent group,” as used herein, means a group selected from thefollowing moieties:

(A) —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, oxo, halogen, unsubstituted alkyl,unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstitutedheterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and

(B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, andheteroaryl, substituted with at least one substituent selected from:

I oxo, —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, halogen, unsubstituted alkyl,unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstitutedheterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and

(ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, andheteroaryl, substituted with at least one substituent selected from:

(a) oxo, —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, halogen, unsubstituted alkyl,unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstitutedheterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and

(b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, orheteroaryl, substituted with at least one substituent selected from oxo,—OH, —NH₂, —SH, —CN, —CF₃, —NO₂, halogen, unsubstituted alkyl,unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstitutedheterocycloalkyl, unsubstituted aryl, and unsubstituted heteroaryl.

A “size-limited substituent” or “ size-limited substituent group,” asused herein means a group selected from all of the substituentsdescribed above for a “substituent group,” wherein each substituted orunsubstituted alkyl is a substituted or unsubstituted C₁-C₂₀ alkyl, eachsubstituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₄-C₈cycloalkyl, and each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 4 to 8 membered heterocycloalkyl.

A “lower substituent” or “ lower substituent group,” as used hereinmeans a group selected from all of the substituents described above fora “substituent group,” wherein each substituted or unsubstituted alkylis a substituted or unsubstituted C₁-C₆ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₅-C₇ cycloalkyl, and each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 5 to 7membered heterocycloalkyl.

The compounds of the present invention may exist as salts. The presentinvention includes such salts. Examples of applicable salt forms includehydrochlorides, hydrobromides, sulfates, methanesulfonates, nitrates,maleates, acetates, citrates, fumarates, tartrates (eg (+)-tartrates,(−)-tartrates or mixtures thereof including racemic mixtures,succinates, benzoates and salts with amino acids such as glutamic acid.These salts may be prepared by methods known to those skilled in art.Also included are base addition salts such as sodium, potassium,calcium, ammonium, organic amino, or magnesium salt, or a similar salt.When compounds of the present invention contain relatively basicfunctionalities, acid addition salts can be obtained by contacting theneutral form of such compounds with a sufficient amount of the desiredacid, either neat or in a suitable inert solvent. Examples of acceptableacid addition salts include those derived from inorganic acids likehydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic,phosphoric, monohydrogen-phosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived organic acids like acetic, propionic,isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric,lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric,tartaric, methanesulfonic, and the like. Also included are salts ofamino acids such as arginate and the like, and salts of organic acidslike glucuronic or galactunoric acids and the like. Certain specificcompounds of the present invention contain both basic and acidicfunctionalities that allow the compounds to be converted into eitherbase or acid addition salts.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents.

The term “pharmaceutically acceptable salts” is meant to include saltsof active compounds which are prepared with relatively nontoxic acids orbases, depending on the particular substituent moieties found on thecompounds described herein. When compounds of the present inventioncontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable base additionsalts include sodium, potassium, calcium, ammonium, organic amino, ormagnesium salt, or a similar salt. When compounds of the presentinvention contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,mono-hydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methane sulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike {see, e.g., Berge et al., Journal of Pharmaceutical Science,66:1-19 (1977)). Certain specific compounds of the present inventioncontain both basic and acidic functionalities that allow the compoundsto be converted into either base or acid addition salts.

In addition to salt forms, the present invention provides compounds,which are in a prodrug form. Prodrugs of the compounds described hereinare those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of the presentinvention. Additionally, prodrugs can be converted to the compounds ofthe present invention by chemical or biochemical methods in an ex vivoenvironment. For example, prodrugs can be slowly converted to thecompounds of the present invention when placed in a transdermal patchreservoir with a suitable enzyme or chemical reagent.

Protecting groups are commonly used in organic synthesis in order toprotect various functional groups, including but not limited to amino,carbonyl, carboxyl, hydroxyl, 1,2-diols and 1,3-diols. One of skill inthe art would know which functional groups would require protection, howto select an appropriate protecting group as well as how to prepare andremove such groups in order to unmask the pre-existing functional group(T. W. Green, P. G. M. Wuts, Protective Groups in Organic Synthesis,Wiley-Interscience, New York, 1999).

Common amino protecting groups include but are not limited to9-fluorenylmethyl carbamate, t-butyl carbamate, benzyl carbamate,acetamide, trifluoroacetamide, phthalimide, benzylamine,triphenylmethylamine, benzylideneamine, p-toluenesulfonamide, and thelike.

Common carbonyl protecting groups include but are not limited todimethyl acetal, 1,3-dioxane, 1,3-dithiane, N,N-dimethylhydrazone, andthe like.

Common carboxyl protecting groups include but are not limited to methylester, t-butyl ester, benzyl ester, S-t-butyl ester,2-alkyl-1,3-oxazoline, and the like.

Common hydroxyl protecting groups include but are not limited tomethoxymethyl ether (MOM), tetrahydropyranyl ether (THP), tert-butylether (t-Bu), allyl ether, benzyl ether, tert-butyldimethylsilyl ether(TBDMS), t-butyldiphenylsilyl ether (TBDPS), acetic acid ester, pivalicacid ester, benzoic acid ester, and the like.

Common 1,2- and 1,3-diol protecting groups include but are not limitedto acetonide, benzylidene acetal, and the like.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical or chiral centers) or double bonds; the enantiomers,racemates, diastereomers, tautomers, geometric isomers, stereoisometricforms that may be defined, in terms of absolute stereochemistry, as (R)—or (S)— or, as (D)- or (L)- for amino acids, and individual isomers areencompassed within the scope of the present invention. The compounds ofthe present invention do not include those which are known in art to betoo unstable to synthesize and/or isolate. The present invention ismeant to include compounds in racemic and optically pure forms.Optically active (R)— and (S)—, or (D)- and (L)-isomers may be preparedusing chiral synthons or chiral reagents, or resolved using conventionaltechniques. When the compounds described herein contain olefinic bondsor other centers of geometric asymmetry, and unless specified otherwise,it is intended that the compounds include both E and Z geometricisomers.

The term “tautomer,” as used herein, refers to one of two or morestructural isomers which exist in equilibrium and which are readilyconverted from one isomeric form to another. It is apparent to oneskilled in the art that certain compounds of this invention may exist intautomeric forms, all such tautomeric forms of the compounds beingwithin the scope of the invention.

Unless otherwise stated, structures depicted herein are also meant toinclude all stereochemical forms of the structure; i.e., the R and Sconfigurations for each asymmetric center. Single stereochemical isomersas well as enantiomeric and diastereomeric mixtures of the presentcompounds are within the scope of the invention.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds which differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of a hydrogen by a deuterium ortritium, or the replacement of a carbon by ¹³C- or ¹⁴C-enriched carbonare within the scope of this invention. The compounds of the presentinvention may also contain unnatural proportions of atomic isotopes atone or more of atoms that constitute such compounds. For example, thecompounds may be radiolabeled with radioactive isotopes, such as forexample tritium (³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). All isotopicvariations of the compounds of the present invention, whetherradioactive or not, are encompassed within the scope of the presentinvention.

The term “subject,” “patient,” or “individual” as used herein inreference to individuals suffering from a disorder, and the like,encompasses mammals and non-mammals. Examples of mammals include, butare not limited to, any member of the Mammalian class: humans, non-humanprimates such as chimpanzees, and other apes and monkey species; farmanimals such as cattle, horses, sheep, goats, swine; domestic animalssuch as rabbits, dogs, and cats; laboratory animals including rodents,such as rats, mice and guinea pigs, and the like. Examples ofnon-mammals include, but are not limited to, birds, fish and the like.In one embodiment of the methods and compositions provided herein, themammal is a human.

The terms “treating” or “treatment” in reference to a particular diseaseincludes prevention of the disease. The terms “treat,” “treating” or“treatment,” and other grammatical equivalents as used herein, includealleviating, abating or ameliorating a disease or condition symptoms,preventing additional symptoms, ameliorating or preventing theunderlying metabolic causes of symptoms, inhibiting the disease orcondition, e.g., arresting the development of the disease or condition,relieving the disease or condition, causing regression of the disease orcondition, relieving a condition caused by the disease or condition, orstopping the symptoms of the disease or condition, and are intended toinclude prophylaxis. The terms further include achieving a therapeuticbenefit and/or a prophylactic benefit. By therapeutic benefit is meanteradication or amelioration of the underlying disorder being treated.Also, a therapeutic benefit is achieved with the eradication oramelioration of one or more of the physiological symptoms associatedwith the underlying disorder such that an improvement is observed in thepatient, notwithstanding that the patient may still be afflicted withthe underlying disorder. In some embodiments, for prophylactic benefit,the compositions are administered to a patient at risk of developing aparticular disease, or to a patient reporting one or more of thephysiological symptoms of a disease, even though a diagnosis of thisdisease may not have been made.

The term “pharmaceutically acceptable” as used herein, refers to amaterial, such as a carrier or diluent, which does not abrogate thebiological activity or properties of the compounds described herein, andis relatively nontoxic, i.e., in other embodiments, the material isadministered to an individual without causing undesirable biologicaleffects or interacting in a deleterious manner with any of thecomponents of the composition in which it is contained.

The term “pharmaceutical composition,” as used herein, refers to abiologically active compound, optionally mixed with at least onepharmaceutically acceptable chemical component, such as, though notlimited to carriers, stabilizers, diluents, dispersing agents,suspending agents, thickening agents, and/or excipients.

The term “carrier” as used herein, refers to relatively nontoxicchemical compounds or agents that facilitate the incorporation of thecompound into cells or tissues.

The term “agonist,” as used herein, refers to a molecule such as thecompound, a drug, an enzyme activator or a hormone modulator whichenhances the activity of another molecule or the activity of a receptorsite.

The term “antagonist,” as used herein, refers to a molecule such as thecompound, a drug, an enzyme inhibitor, or a hormone modulator, whichdiminishes, or prevents the action of another molecule or the activityof a receptor site.

The term “modulate,” as used herein, means to interact with a targeteither directly or indirectly so as to alter the activity of the target,including, by way of example only, to enhance the activity of thetarget, to inhibit the activity of the target, to limit the activity ofthe target, or to extend the activity of the target.

The term “modulator,” as used herein, refers to a molecule thatinteracts with a target either directly or indirectly. The interactionsinclude, but are not limited to, the interactions of an agonist and anantagonist.

The term “pharmaceutically acceptable derivative or prodrug” as usedherein, refers to any pharmaceutically acceptable salt, ester, salt ofan ester or other derivative of the compound of Formula I, which, uponadministration to a recipient, is capable of providing, either directlyor indirectly, the compound disclosed herein or a pharmaceuticallyactive metabolite or residue thereof. Particularly favored derivativesor prodrugs are those that increase the bioavailability of the compoundsdescribed herein when such compounds are administered to a patient(e.g., by allowing orally administered compound to be more readilyabsorbed into blood) or which enhance delivery of the parent compound toa biological compartment (e.g., the brain or lymphatic system).

The terms “enhance” or “enhancing,” as used herein, means to increase orprolong either in potency or duration a desired effect. Thus, in regardto enhancing the effect of therapeutic agents, the term “enhancing”refers to the ability to increase or prolong, either in potency orduration, the effect of other therapeutic agents on a system. An“enhancing-effective amount,” as used herein, refers to an amountadequate to enhance the effect of another therapeutic agent in a desiredsystem.

The term “metabolite,” as used herein, refers to a derivative of thecompound which is formed when the compound is metabolized.

The term “active metabolite,” as used herein, refers to a biologicallyactive derivative of the compound that is formed when the compound ismetabolized.

The term “metabolized,” as used herein, refers to the sum of theprocesses (including, but not limited to, hydrolysis reactions andreactions catalyzed by enzymes) by which a particular substance ischanged by an organism. Thus, in some embodiments, enzymes producespecific structural alterations to the compound. For example, cytochromeP450 catalyzes a variety of oxidative and reductive reactions whileuridine diphosphate glucuronyltransferases catalyze the transfer of anactivated glucuronic-acid molecule to aromatic alcohols, aliphaticalcohols, carboxylic acids, amines and free sulphydryl groups. Furtherinformation on metabolism may be obtained from The Pharmacological Basisof Therapeutics, 9th Edition, McGraw-Hill (1996).

The term “genome” includes but is not limited to the human genome. Othergenomes include for example: viruses, for example, bacteriophage MS2,SV40, phage Φ-X17, HIV, phage λ, and mimivirus; bacterium, for example,Haemophilus influenzae, Carsonella ruddi, buchnera aphidicola,wigglesworthia glossinidia, and escherichia coli; amoeboid, for example,polychaos dubium (“Amoeba” dubia); plant, for example, arabidopsisthaliana, genlisea margaretae, fritillaria assyrica, populustrichocarpa, and paris japonica (Japanese-native, pale-petal); Moss, forexample, physcomitrella patens; yeast, for example, saccharomycescerevisiae; fungus, for example, aspergillus nidulans;nematode, forexample, caenorhabditis elegans and pratylenchus coffeae; insect, forexample, drosophila melanogaster (fruit fly), bombyx mori (silk moth),apis mellifera (honey bee), solenopsis invicta (fire ant), tetraodonnigroviridis (type of puffer fish); mammal, for example, homo sapiens(humans); and fish, for example, protopterus aethiopicus (marbledlungfish) and the like.Hybrid Cyclic LibrariesIn one embodiment thedisclosure provides a compound of Formula I:

or a pharmaceutically acceptable salt or solvate thereof, wherein:

is a single or double bond;

X₁ is O or NR⁶;

Y is —C(O)— or

X₂ is (CH₂)_(m), O, or NR⁶;

Z is

W is O, CH, CH₂, CR⁴, or CR⁵;

L₁ and L₂ are each independently a direct bond, substituted orunsubstituted —(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)O(C₁-C₆)alkyl-, substituted or unsubstituted —(CH₂)_(n)C(O)—,substituted or unsubstituted —(CH₂)_(n)C(O)(C₁-C₆)alkyl-, substituted orunsubstituted —(CH₂)_(n)C(O)O(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)NH(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)S(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₁-C₆)alkyl-, substituted or unsubstituted—(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)O(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)C(O)O(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)NH(C₁-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)S(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkenyl-, substituted or unsubstituted—(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)O(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)C(O)O(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)NH(C₁-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)S(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkynyl-, wherein each alkyl, alkenyl andalkynyl group may be optionally substituted with alkyl, alkoxy, amino,carboxyl, cyano, nitro, or trifluoromethyl;

each m is independently an integer selected from 0, 1, 2, 3, 4, 5, and6;

each n is independently an integer selected from 0, 1, 2, 3, 4, 5, and6;

R¹ is hydrogen, hydroxyl, or OPG, wherein PG is a protecting group, or

R¹ is

wherein

is a resin;

R² is hydrogen, hydroxyl, or alkoxy;

R³ is hydrogen or alkyl;

R⁴ and R⁵ are each independently hydrogen, hydroxy, alkyl, alkoxy, orOPG, wherein PG is a protecting group;

R⁶ is hydrogen or alkyl;

wherein the Effector Domain has Formula II:

wherein:

R⁷, R⁹, R¹¹, R¹³, and R¹⁵ are each independently hydrogen or alkyl;

R⁸, R¹⁰, R¹², and R¹⁴ are each independently hydrogen, halogen, amino,cyano, nitro, trifluoromethyl, substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted perfluoroalkyl, substituted or unsubstituted alkoxy,substituted or unsubstituted alkylamino, substituted or unsubstitutedalkylthio, substituted or unsubstituted aryl, substituted orunsubstituted alkylaryl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted heteroaryl, substituted or unsubstituted heteroalkylaryl,(CH₂)_(n)CN, (CH₂)_(n)CF₃, (CH₂)_(n)C₂F₅, (CH₂)_(n)OR¹⁶,(CH₂)_(n)C(O)R¹⁶, (CH₂)_(n)C(O)OR¹⁶, (CH₂)_(n)OC(O)R¹⁶,(CH₂)_(n)NR¹⁷R¹⁸, (CH₂)_(n)C(O)NR¹⁷R¹⁸, (CH₂)_(n)N¹⁹RC(O)R¹⁶,(CH₂)_(n)N¹⁹RC(O)OR¹⁶, (CH₂)_(n)NR¹⁹C(O)NR¹⁷R¹⁸, (CH₂)_(n)SR¹⁶,(CH₂)_(n)S(O)_(j)NR¹⁷R¹⁸, (CH₂)_(n)N¹⁹RS(O)_(j)R¹⁶, or—(CH₂)_(n)NR¹⁹S(O)_(j)NR¹⁷R¹⁸;

n is an integer selected from 0, 1, 2, 3, 4, 5, and 6;

j is an integer selected from 0, 1, and 2;

R¹⁶, R¹⁷, R¹⁸, and R¹⁹ are each independently hydrogen, halogen, amino,cyano, nitro, trifluoromethyl, alkyl, alkenyl, alkynyl, cycloalkyl,perfluoroalkyl, alkoxy, alkylamino, alkylthio, aryl, alkylaryl,heteroalkyl, heterocycloalkyl, heteroaryl, or heteroalkylaryl, or

R¹⁶ and R¹⁹ are as described above, and R¹⁷ and R¹⁸, together with the Natom to which they are attached, form a substituted or unsubstituted 5-,6-, or 7-membered heterocycloalkyl or a substituted or unsubstituted5-membered heteroaryl,

wherein each of the above groups listed for R⁸, R¹⁰, R¹², and R¹⁴ may beoptionally independently substituted with 1 to 3 groups selected fromhalogen, amino, cyano, nitro, trifluoromethyl, alkyl, alkenyl, alkynyl,cycloalkyl, perfluoroalkyl, alkoxy, alkylamino, alkylthio, aryl,alkylaryl, heteroalkyl, heterocycloalkyl, heteroaryl, heteroalkylaryl,(CH₂)_(n)CN, (CH₂)_(n)CF₃, (CH₂)_(n)C₂F₅, (CH₂)_(n)OR¹⁶,(CH₂)_(n)C(O)R¹⁶, (CH₂)_(n)C(O)OR¹⁶, (CH₂)_(n)OC(O)R¹⁶,(CH₂)_(n)NR¹⁷R¹⁸, (CH₂)_(n)C(O)NR¹⁷R¹⁸, (CH₂)_(n)N¹⁹RC(O)R¹⁶,(CH₂)_(n)N¹⁹RC(O)OR¹⁶, (CH₂)_(n)NR¹⁹C(O)NR¹⁷R¹⁸, (CH₂)_(n)SR¹⁶,(CH₂)_(n)S(O)_(j)NR¹⁷R¹⁸, (CH₂)_(n)N¹⁹RS(O)_(j)R¹⁶, and—(CH₂)_(n)NR¹⁹S(O)_(j)NR¹⁷R¹⁸.

In another embodiment, the disclosure provides a compound of Formula I,wherein:

X is O or NR⁶;

L₁ and L₂ are each independently —(C₁-C₆)alkyl-,—(CH₂)_(n)O(C₁-C₆)alkyl-, —(CH₂)_(n)C(O)(C₁-C₆)alkyl-,—(CH₂)_(n)C(O)O(C₁-C₆)alkyl-, —(CH₂)_(n)NH(C₁-C₆)alkyl-,—(CH₂)_(n)C(O)NH(C₁-C₆)alkyl-, —(CH₂)_(n)S(C₁-C₆)alkyl-,—(CH₂)_(n)C(O)(CH₂)_(n)S(C₁-C₆)alkyl-, —(C₂-C₆)alkenyl-,—(CH₂)_(n)O(C₂-C₆)alkenyl-, —(CH₂)_(n)C(O)(C₂-C₆)alkenyl-,—(CH₂)_(n)C(O)O(C₂-C₆)alkenyl-, —(CH₂)_(n)NH(C₁-C₆)alkenyl-,—(CH₂)_(n)C(O)NH(C₂-C₆)alkenyl-, —(CH₂)_(n)S(C₂-C₆)alkenyl-,—(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkenyl-, wherein each alkyl and alkenylgroup may be substituted with alkyl, alkoxy, or carboxyl;

n is an integer selected from 0, 1, 2, 3, 4, 5, and 6;

R¹ is hydrogen, hydroxyl, or OPG, wherein PG is a silyl protectinggroup, or

R¹ is

wherein

is a resin;

R² is hydroxyl or alkoxy;

R³ is hydrogen or alkyl;

R⁴ and R⁵ are each independently hydrogen, alkyl, alkoxy, or OPG,wherein PG is a silyl protecting group;

R⁶ is hydrogen;

R⁷, R⁹, R¹¹, R¹³, and R¹⁵ are each independently hydrogen or CH₃;

R⁸, R¹⁰, R¹², and R¹⁴ are each independently substituted orunsubstituted cyclopropyl, substituted or unsubstituted cyclobutyl,substituted or unsubstituted cyclopentyl, substituted or unsubstitutedcyclohexyl, substituted or unsubstituted cycloheptyl, or substituted orunsubstituted cyclooctyl; or

R⁸, R¹⁰, R¹², and R¹⁴ are each independently substituted orunsubstituted tetrahydrofuranyl, substituted or unsubstitutedtetrahydrothiophenyl, substituted or unsubstituted pyrrolidinyl,substituted or unsubstituted 1,3-dioxolanyl, substituted orunsubstituted pyrazolidinyl, substituted or unsubstitutedimidazolidinyl, substituted or unsubstituted 1,4-dioxanyl, substitutedor unsubstituted piperidinyl, substituted or unsubstituted piperazinyl,substituted or unsubstituted morpholinyl, substituted or unsubstitutedthiomorpholinyl, or substituted or unsubstituted 1,4-dithianyl; or

R⁸, R¹⁰, R¹², and R¹⁴ are each independently substituted orunsubstituted phenyl, substituted or unsubstituted benzyl, substitutedor unsubstituted naphthylenyl, or substituted or unsubstituted biphenyl;or

R⁸, R¹⁰, R¹², and R¹⁴ are each independently substituted orunsubstituted furanyl, substituted or unsubstituted thiophenyl,substituted or unsubstituted pyrrolyl, substituted or unsubstitutedpyrazolyl, substituted or unsubstituted imidazolyl, substituted orunsubstituted triazolyl, substituted or unsubstituted isoxazolyl,substituted or unsubstituted oxazolyl, substituted or unsubstitutedthiazolyl, substituted or unsubstituted isothiazolyl, substituted orunsubstituted pyridinyl, substituted or unsubstituted pyridizanyl,substituted or unsubstituted pyrimidinyl, substituted or unsubstitutedtriazinyl, substituted or unsubstituted benzofuranyl, substituted orunsubstituted benzo(b)thiophenyl, substituted or unsubstituted indolyl,substituted or unsubstituted benzimidazolyl, substituted orunsubstituted indazolyl, substituted or unsubstituted benzisoxazolyl,substituted or unsubstituted benzoxazolyl, substituted or unsubstitutedbenzothiazolyl, substituted or unsubstituted quinolinyl, substituted orunsubstituted isoquinolinyl, substituted or unsubstituted quinazolinyl,substituted or unsubstituted quinoxalinyl, or substituted orunsubstituted naphthyridinyl.

In another embodiment, the disclosure provides a compound of Formula I,wherein:

L₁ and L₂ are each independently —(C₁-C₆)alkyl-,—(CH₂)_(n)O(C₁-C₆)alkyl-, —(CH₂)_(n)C(O)(C₁-C₆)alkyl-,—(CH₂)_(n)NH(C₁-C₆)alkyl-, —(CH₂)_(n)C(O)NH(C₁-C₆)alkyl-,—(C₂-C₆)alkenyl-, —(CH₂)_(n)O(C₂-C₆)alkenyl-,—(CH₂)_(n)C(O)(C₂-C₆)alkenyl-, —(CH₂)_(n)NH(C₁-C₆)alkenyl-,—(CH₂)_(n)C(O)NH(C₂-C₆)alkenyl-, wherein each alkyl and alkenyl groupmay be substituted with alkyl, alkoxy, or carboxyl;

n is an integer selected from 0, 1, 2, 3, 4, 5, and 6;

R¹ is hydrogen, hydroxyl or OPG, wherein PG is a tert-butyldimethylsilylprotecting group, or

R¹ is

wherein

is Wang resin;

R⁴ and R⁵ are each independently hydrogen, hydroxy, alkyl, alkoxy, orOPG, wherein PG is a tert-butyldimethylsilyl protecting group;

R⁷, R⁹, R¹¹, R¹³, and R¹⁵ are each independently hydrogen;

R⁸, R¹⁰, R¹², and R¹⁴ are each independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted benzyl, substituted orunsubstituted pyrrolidinyl, substituted or unsubstituted indolyl,(CH₂)_(n)OR⁵, (CH₂)_(n)C(O)NR⁶R⁷, or (CH₂)_(n)SR⁵; and

R¹⁶, R¹⁷, and R¹⁸ are each independently hydrogen or (C₁-C₆)alkyl.

In another embodiment, the disclosure provides a compound of Formula I,wherein:

L₁ and L₂ are each independently —(C₁-C₆)alkyl-, —O(C₁-C₆)alkyl-,—C(O)(C₁-C₆)alkyl-, —(CH₂)_(n)C(O)NH(C₁-C₆)alkyl-, —(C₂-C₆)alkenyl-,—O(C₂-C₆)alkenyl-, —C(O)(C₂-C₆)alkenyl-,—(CH₂)_(n)C(O)NH(C₂-C₆)alkenyl-, wherein each alkyl and alkenyl groupmay be substituted with alkyl, alkoxy, or carboxyl;

n is an integer selected from 0, 1, 2, 3, 4, 5, and 6;

R⁸, R¹⁰, R¹², and R¹⁴ are each independently H, CH₃, CH₂OH, CH₂SH,CH(OH)CH₃, CH₂C(O)NH₂, CH₂CH₂C(O)NH₂, CH₂CH₂SCH₃, CH₂CH(CH₃)₂,CH(CH₃)CH₂CH₃, CH₂C₆C₅,

In another embodiment, the disclosure provides a compound of Formula I,wherein:

L₁ and L₂ are each independently —OCH₂CH₂—, —CH₂C(O)—, —CH₂CH₂C(O)—,—C(O)NHCH₂CH₂, —CH₂CH═CHCH₂—, —OCH₂CH═CHCH₂CH₂—, —OCH₂CH═CHCH₂CH(CO2H)—,—CH₂C(O)NHCH₂CH₂—, or CH₂CH(OCH₃)═C(CH₃)CH₂CH_(2;) and

R⁸, R¹⁰, R¹², and R¹⁴ are each independently the sidechain of the aminoacid alanine, asparagine, cysteine, glutamine, glycine, isoleucine,leucine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, or valine.

In another embodiment, the disclosure provides a compound of Formula I,wherein the compound has Formulae II, III, IV, V, or VI:

In another embodiment, the disclosure provides a compound of Formula I,wherein the compound has Formulae VII, VIII, IX, or X:

In another embodiment, the disclosure provides methods for treatingcancer in a patient in need thereof by administering the compound ofFormula I to the patient.

In another embodiment, the disclosure provides methods for suppressingthe immune system in a patient in need thereof by administering thecompound of Formula I to the patient.

In another embodiment, the disclosure provides methods for preparing acompound of Formula I:

or a pharmaceutically acceptable salt or solvate thereof, wherein:

is a single or double bond;

X₁ is O or NR⁶;

Y is —C(O)— or

X₂ is (CH₂)_(m), O, or NR⁶;

Z is

W is O, CH, CH₂, CR⁴, or CR⁵;

L₁ and L₂ are each independently a direct bond, substituted orunsubstituted —(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)O(C₁-C₆)alkyl-, substituted or unsubstituted —(CH₂)_(n)C(O)—,substituted or unsubstituted —(CH₂)_(n)C(O)(C₁-C₆)alkyl-, substituted orunsubstituted —(CH₂)_(n)C(O)O(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)NH(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)S(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₁-C₆)alkyl-, substituted or unsubstituted—(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)O(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)C(O)O(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)NH(C₁-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)S(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkenyl-, substituted or unsubstituted—(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)O(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)C(O)O(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)NH(C₁-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)S(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkynyl-, wherein each alkyl, alkenyl andalkynyl group may be optionally substituted with alkyl, alkoxy, amino,carboxyl, cyano, nitro, or trifluoromethyl;

each m is independently an integer selected from 0, 1, 2, 3, 4, 5, and6;

each n is independently an integer selected from 0, 1, 2, 3, 4, 5, and6;

R¹ is hydrogen, hydroxyl, or OPG, wherein PG is a protecting group, or

R¹ is

wherein

is a resin;

R² is hydrogen, hydroxyl, or alkoxy;

R³ is hydrogen or alkyl;

R⁴ and R⁵ are each independently hydrogen, hydroxy, alkyl, alkoxy, orOPG, wherein PG is a protecting group;

R⁶ is hydrogen or alkyl;

wherein the Effector Domain has Formula II:

wherein:

R⁷, R⁹, R¹¹, R¹³, and R¹⁵ are each independently hydrogen or alkyl;

R⁸, R¹⁰, R¹², and R¹⁴ are each independently hydrogen, halogen, amino,cyano, nitro, trifluoromethyl, substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted perfluoroalkyl, substituted or unsubstituted alkoxy,substituted or unsubstituted alkylamino, substituted or unsubstitutedalkylthio, substituted or unsubstituted aryl, substituted orunsubstituted alkylaryl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted heteroaryl, substituted or unsubstituted heteroalkylaryl,(CH₂)_(n)CN, (CH₂)_(n)CF₃, (CH₂)_(n)C₂F₅, (CH₂)_(n)OR¹⁶,(CH₂)_(n)C(O)R¹⁶, (CH₂)_(n)C(O)OR¹⁶, (CH₂)_(n)OC(O)R¹⁶,(CH₂)_(n)NR¹⁷R¹⁸, (CH₂)_(n)C(O)NR¹⁷R¹⁸, (CH₂)_(n)N¹⁹RC(O)R¹⁶,(CH₂)_(n)N¹⁹RC(O)OR¹⁶, (CH₂)_(n)NR¹⁹C(O)NR¹⁷R¹⁸, (CH₂)_(n)SR¹⁶,(CH₂)_(n)S(O)_(j)NR¹⁷R¹⁸, (CH₂)_(n)N¹⁹RS(O)_(j)R¹⁶, or—(CH₂)_(n)NR¹⁹S(O)_(j)NR¹⁷R¹⁸;

n is an integer selected from 0, 1, 2, 3, 4, 5, and 6;

j is an integer selected from 0, 1, and 2;

R¹⁶, R¹⁷, R¹⁸, and R¹⁹ are each independently hydrogen, halogen, amino,cyano, nitro, trifluoromethyl, alkyl, alkenyl, alkynyl, cycloalkyl,perfluoroalkyl, alkoxy, alkylamino, alkylthio, aryl, alkylaryl,heteroalkyl, heterocycloalkyl, heteroaryl, or heteroalkylaryl, or

R¹⁶ and R¹⁹ are as described above, and R¹⁷ and R¹⁸, together with the Natom to which they are attached, form a substituted or unsubstituted 5-,6-, or 7-membered heterocycloalkyl or a substituted or unsubstituted5-membered heteroaryl,

wherein each of the above groups listed for R⁸, R¹⁰, R¹², and R¹⁴ may beoptionally independently substituted with 1 to 3 groups selected fromhalogen, amino, cyano, nitro, trifluoromethyl, alkyl, alkenyl, alkynyl,cycloalkyl, perfluoroalkyl, alkoxy, alkylamino, alkylthio, aryl,alkylaryl, heteroalkyl, heterocycloalkyl, heteroaryl, heteroalkylaryl,(CH₂)_(n)CN, (CH₂)_(n)CF₃, (CH₂)_(n)C₂F₅, (CH₂)_(n)OR¹⁶,(CH₂)_(n)C(O)R¹⁶, (CH₂)_(n)C(O)OR¹⁶, (CH₂)_(n)OC(O)R¹⁶,(CH₂)_(n)NR¹⁷R¹⁸, (CH₂)_(n)C(O)NR¹⁷R¹⁸, (CH₂)_(n)N¹⁹RC(O)R¹⁶,(CH₂)_(n)N¹⁹RC(O)OR¹⁶, (CH₂)_(n)NR¹⁹C(O)NR¹⁷R¹⁸, (CH₂)_(n)SR¹⁶,(CH₂)_(n)S(O)_(j)NR¹⁷R¹⁸, (CH₂)_(n)N¹⁹RS(O)_(j)R¹⁶, and—(CH₂)_(n)NR¹⁹S(O)_(j)NR¹⁷R¹⁸;

the method comprising the steps of: a) coupling and cyclizing a compoundof Formula XI with the Effector Domain to provide the compound ofFormula I:

In another embodiment, the disclosure provides methods for preparing acompound of Formula I, wherein the reagentsbenzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate(PyBOP), N,N-diisopropylethyl-amine (DIPEA), and N-methylpyrrolidine(NIMP) are used in coupling.

In another embodiment, the disclosure provides methods for preparing acompound of Formula I, wherein the reagentbenzylidene[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene]dichloro(tricyclohexylphosphine)rutheniumis used in cyclizing.

In another embodiment, the disclosure provides a compound of Formula I,prepared by the methods described herein.

In another embodiment, the disclosure provides methods for identifyingcompounds from a library of compounds that binds to a protein encoded ina genome in complex with FKBP, by:

a) screening a hybrid combinatorial peptide or non-peptide library ofcompounds that includes the FKBP-binding domain (FKBP) of the naturalproduct rapamycin or FK506 against the proteins encoded in a genomeusing a protein chip;

b) detecting the binding of a compound to a protein on the chip usingthe anti-V5 antibody together with a fluorescently tagged secondaryantibody;

c) recording the fluorescence pattern of the protein chip on a chipreader;

d) identifying the proteins based on the physical location of thefluorescent spots on the chip; and

e) determining the function of the protein based on its perturbedbiochemical and cellular functions.

In another embodiment, the disclosure provides methods for identifyingcompounds from a library of compounds that binds to a protein encoded ina genome in complex with FKBP, wherein the genome is the human genome;and the library of compounds has Formula I:

or a pharmaceutically acceptable salt or solvate thereof, wherein:

is a single or double bond;

X₁ is O or NR⁶;

Y is —C(O)— or

X₂ is (CH₂)_(m), O, or NR⁶;

Z is

W is O, CH, CH₂, CR⁴, or CR⁵;

L₁ and L₂ are each independently a direct bond, substituted orunsubstituted —(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)O(C₁-C₆)alkyl-, substituted or unsubstituted —(CH₂)_(n)C(O)—,substituted or unsubstituted —(CH₂)_(n)C(O)(C₁-C₆)alkyl-, substituted orunsubstituted —(CH₂)_(n)C(O)O(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)NH(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)S(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₁-C₆)alkyl-, substituted or unsubstituted—(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)O(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)C(O)O(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)NH(C₁-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)S(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkenyl-, substituted or unsubstituted—(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)O(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)C(O)O(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)NH(C₁-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)S(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkynyl-, wherein each alkyl, alkenyl andalkynyl group may be optionally substituted with alkyl, alkoxy, amino,carboxyl, cyano, nitro, or trifluoromethyl;

each m is independently an integer selected from 0, 1, 2, 3, 4, 5, and6;

each n is independently an integer selected from 0, 1, 2, 3, 4, 5, and6;

R¹ is hydrogen, hydroxyl, or OPG, wherein PG is a protecting group, or

R¹ is

wherein

is a resin;

R² is hydrogen, hydroxyl, or alkoxy;

R³ is hydrogen or alkyl;

R⁴ and R⁵ are each independently hydrogen, hydroxy, alkyl, alkoxy, orOPG, wherein PG is a protecting group;

R⁶ is hydrogen or alkyl;

wherein the Effector Domain has Formula II:

wherein:

R⁷, R⁹, R¹¹, R¹³, and R¹⁵ are each independently hydrogen or alkyl;

R⁸, R¹⁰, R¹², and R¹⁴ are each independently hydrogen, halogen, amino,cyano, nitro, trifluoromethyl, substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted perfluoroalkyl, substituted or unsubstituted alkoxy,substituted or unsubstituted alkylamino, substituted or unsubstitutedalkylthio, substituted or unsubstituted aryl, substituted orunsubstituted alkylaryl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted heteroaryl, substituted or unsubstituted heteroalkylaryl,(CH₂)_(n)CN, (CH₂)_(n)CF₃, (CH₂)_(n)C₂F₅, (CH₂)_(n)OR¹⁶,(CH₂)_(n)C(O)R¹⁶, (CH₂)_(n)C(O)OR¹⁶, (CH₂)_(n)OC(O)R¹⁶,(CH₂)_(n)NR¹⁷R¹⁸, (CH₂)_(n)C(O)NR¹⁷R¹⁸, (CH₂)_(n)N¹⁹RC(O)R¹⁶,(CH₂)_(n)N¹⁹RC(O)OR¹⁶, (CH₂)_(n)NR¹⁹C(O)NR¹⁷R¹⁸, (CH₂)_(n)SR¹⁶,(CH₂)_(n)S(O)_(j)NR¹⁷R¹⁸, (CH₂)_(n)N¹⁹RS(O)_(j)R¹⁶, or—(CH₂)_(n)NR¹⁹S(O)_(j)NR¹⁷R¹⁸;

n is an integer selected from 0, 1, 2, 3, 4, 5, and 6;

j is an integer selected from 0, 1, and 2;

R¹⁶, R¹⁷, R¹⁸, and R¹⁹ are each independently hydrogen, halogen, amino,cyano, nitro, trifluoromethyl, alkyl, alkenyl, alkynyl, cycloalkyl,perfluoroalkyl, alkoxy, alkylamino, alkylthio, aryl, alkylaryl,heteroalkyl, heterocycloalkyl, heteroaryl, or heteroalkylaryl, or

R¹⁶ and R¹⁹ are as described above, and R¹⁷ and R¹⁸, together with the Natom to which they are attached, form a substituted or unsubstituted 5-,6-, or 7-membered heterocycloalkyl or a substituted or unsubstituted5-membered heteroaryl,

wherein each of the above groups listed for R⁸, R¹⁰, R¹², and R¹⁴ may beoptionally independently substituted with 1 to 3 groups selected fromhalogen, amino, cyano, nitro, trifluoromethyl, alkyl, alkenyl, alkynyl,cycloalkyl, perfluoroalkyl, alkoxy, alkylamino, alkylthio, aryl,alkylaryl, heteroalkyl, heterocycloalkyl, heteroaryl, heteroalkylaryl,(CH₂)_(n)CN, (CH₂)_(n)CF₃, (CH₂)_(n)C₂F₅, (CH₂)_(n)OR¹⁶,(CH₂)_(n)C(O)R¹⁶, (CH₂)_(n)C(O)OR¹⁶, (CH₂)_(n)OC(O)R¹⁶,(CH₂)_(n)NR¹⁷R¹⁸, (CH₂)_(n)C(O)NR¹⁷R¹⁸, (CH₂)_(n)N¹⁹RC(O)R¹⁶,(CH₂)_(n)N¹⁹RC(O)OR¹⁶, (CH₂)_(n)NR¹⁹C(O)NR¹⁷R¹⁸, (CH₂)_(n)SR¹⁶,(CH₂)_(n)S(O)_(j)NR¹⁷R¹⁸, (CH₂)_(n)N¹⁹RS(O)_(j)R¹⁶, and—(CH₂)_(n)NR¹⁹S(O)_(j)NR¹⁷R¹⁸.

In another embodiment, the disclosure provides methods for determiningthe function of a protein encoded in a genome, wherein the compound ofFormula I:

X is O or NR⁶;

L₁ and L₂ are each independently —(C₁-C₆)alkyl-,—(CH₂)_(n)O(C₁-C₆)alkyl-, —(CH₂)_(n)C(O)(C₁-C₆)alkyl-,—(CH₂)_(n)C(O)O(C₁-C₆)alkyl-, —(CH₂)_(n)NH(C₁-C₆)alkyl-,—(CH₂)_(n)C(O)NH(C₁-C₆)alkyl-, —(CH₂)_(n)S(C₁-C₆)alkyl-,—(CH₂)_(n)C(O)(CH₂)_(n)S(C₁-C₆)alkyl-, —(C₂-C₆)alkenyl-,—(CH₂)_(n)O(C₂-C₆)alkenyl-, —(CH₂)_(n)C(O)(C₂-C₆)alkenyl-,—(CH₂)_(n)C(O)O(C₂-C₆)alkenyl-, —(CH₂)_(n)NH(C₁-C₆)alkenyl-,—(CH₂)_(n)C(O)NH(C₂-C₆)alkenyl-, —(CH₂)_(n)S(C₂-C₆)alkenyl-,—(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkenyl-, wherein each alkyl and alkenylgroup may be substituted with alkyl, alkoxy, or carboxyl;

n is an integer selected from 0, 1, 2, 3, 4, 5, and 6;

R¹ is hydrogen, hydroxyl, or OPG, wherein PG is a silyl protectinggroup, or

R¹ is

wherein

is a resin;

R² is hydroxyl or alkoxy;

R³ is hydrogen or alkyl;

R⁴ and R⁵ are each independently hydrogen, alkyl, alkoxy, or OPG,wherein PG is a silyl protecting group;

R⁶ is hydrogen;

R⁷, R⁹, R¹¹, R¹³, and R¹⁵ are each independently hydrogen or CH₃;

R⁸, R¹⁰, R¹², and R¹⁴ are each independently substituted orunsubstituted cyclopropyl, substituted or unsubstituted cyclobutyl,substituted or unsubstituted cyclopentyl, substituted or unsubstitutedcyclohexyl, substituted or unsubstituted cycloheptyl, or substituted orunsubstituted cyclooctyl; or

R⁸, R¹⁰, R¹², and R¹⁴ are each independently substituted orunsubstituted tetrahydrofuranyl, substituted or unsubstitutedtetrahydrothiophenyl, substituted or unsubstituted pyrrolidinyl,substituted or unsubstituted 1,3-dioxolanyl, substituted orunsubstituted pyrazolidinyl, substituted or unsubstitutedimidazolidinyl, substituted or unsubstituted 1,4-dioxanyl, substitutedor unsubstituted piperidinyl, substituted or unsubstituted piperazinyl,substituted or unsubstituted morpholinyl, substituted or unsubstitutedthiomorpholinyl, or substituted or unsubstituted 1,4-dithianyl; or

R⁸, R¹⁰, R¹², and R¹⁴ are each independently substituted orunsubstituted phenyl, substituted or unsubstituted benzyl, substitutedor unsubstituted naphthylenyl, or substituted or unsubstituted biphenyl;or

R⁸, R¹⁰, R¹², and R¹⁴ are each independently substituted orunsubstituted furanyl, substituted or unsubstituted thiophenyl,substituted or unsubstituted pyrrolyl, substituted or unsubstitutedpyrazolyl, substituted or unsubstituted imidazolyl, substituted orunsubstituted triazolyl, substituted or unsubstituted isoxazolyl,substituted or unsubstituted oxazolyl, substituted or unsubstitutedthiazolyl, substituted or unsubstituted isothiazolyl, substituted orunsubstituted pyridinyl, substituted or unsubstituted pyridizanyl,substituted or unsubstituted pyrimidinyl, substituted or unsubstitutedtriazinyl, substituted or unsubstituted benzofuranyl, substituted orunsubstituted benzo(b)thiophenyl, substituted or unsubstituted indolyl,substituted or unsubstituted benzimidazolyl, substituted orunsubstituted indazolyl, substituted or unsubstituted benzisoxazolyl,substituted or unsubstituted benzoxazolyl, substituted or unsubstitutedbenzothiazolyl, substituted or unsubstituted quinolinyl, substituted orunsubstituted isoquinolinyl, substituted or unsubstituted quinazolinyl,substituted or unsubstituted quinoxalinyl, or substituted orunsubstituted naphthyridinyl.

In another embodiment, the disclosure provides methods for identifyingcompounds from a library of compounds that binds to a protein encoded ina genome in complex with FKBP, wherein the compound of Formula I:

L₁ and L₂ are each independently —(C₁-C₆)alkyl-,—(CH₂)_(n)O(C₁-C₆)alkyl-, —(CH₂)_(n)C(O)(C₁-C₆)alkyl-,—(CH₂)_(n)NH(C₁-C₆)alkyl-, —(CH₂)_(n)C (O)NH(C₁-C₆)alkyl-,—(C₂-C₆)alkenyl-, —(CH₂)_(n)O(C₂-C₆)alkenyl-,—(CH₂)_(n)C(O)(C₂-C₆)alkenyl-, —(CH₂)_(n)NH(C₁-C₆)alkenyl-,—(CH₂)_(n)C(O)NH(C₂-C₆)alkenyl-, wherein each alkyl and alkenyl groupmay be substituted with alkyl, alkoxy, or carboxyl;

n is an integer selected from 0, 1, 2, 3, 4, 5, and 6;

R¹ is hydrogen, hydroxyl or OPG, wherein PG is a tert-butyldimethylsilylprotecting group, or

R¹ is

wherein

is Wang resin;

R⁴ and R⁵ are each independently hydrogen, hydroxy, alkyl, alkoxy, orOPG, wherein PG is a tert-butyldimethylsilyl protecting group;

R⁷, R⁹, R¹¹, R¹³, and R¹⁵ are each independently hydrogen;

R⁸, R¹⁰, R¹², and R¹⁴ are each independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted benzyl, substituted orunsubstituted pyrrolidinyl, substituted or unsubstituted indolyl,(CH₂)_(n)OR⁵, (CH₂)_(n)C(O)NR⁶R⁷, or (CH₂)_(n)SR⁵; and

R¹⁶, R¹⁷, and R¹⁸ are each independently hydrogen or (C₁-C₆)alkyl.

In another embodiment, the disclosure provides methods for identifyingcompounds from a library of compounds that binds to a protein encoded ina genome in complex with FKBP, wherein the compound of Formula I:

L₁ and L₂ are each independently —(C₁-C₆)alkyl-, —O(C₁-C₆)alkyl-,—C(O)(C₁-C₆)alkyl-, —(CH₂)_(n)C(O)NH(C₁-C₆)alkyl-, —(C₂-C₆)alkenyl-,—O(C₂-C₆)alkenyl-, —C(O)(C₂-C₆)alkenyl-,—(CH₂)_(n)C(O)NH(C₂-C₆)alkenyl-, wherein each alkyl and alkenyl groupmay be substituted with alkyl, alkoxy, or carboxyl;

n is an integer selected from 0, 1, 2, 3, 4, 5, and 6;

R⁸, R¹⁰, R¹², and R¹⁴ are each independently H, CH₃, CH₂OH, CH₂SH,CH(OH)CH₃, CH₂C(O)NH₂, CH₂CH₂C(O)NH₂, CH₂CH₂SCH₃, CH₂CH(CH₃)₂,CH(CH₃)CH₂CH₃, CH₂C₆C₅,

In another embodiment, the disclosure provides methods for identifyingcompounds from a library of compounds that binds to a protein encoded ina genome in complex with FKBP, wherein the compound of Formula I:

L₁ and L₂ are each independently OCH₂CH₂—, —CH₂C(O)—, —CH₂CH₂C(O)—,—C(O)NHCH₂CH₂, —CH₂CH═CHCH₂—, —OCH₂CH═CHCH₂CH₂—, —OCH₂CH═CHCH₂CH(CO2H)—,—CH₂C(O)NHCH₂CH₂—, or CH₂CH(OCH₃)═C(CH₃)CH₂CH_(2;) and

R⁸, R¹⁰, R¹², and R¹⁴ are each independently the sidechain of the aminoacid alanine, asparagine, cysteine, glutamine, glycine, isoleucine,leucine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, or valine.

In another embodiment, the disclosure provides methods for generating alead compound as a high-affinity ligand of the FKBP isoforms, the methodcomprising the steps of:

a) screening a hybrid combinatorial peptide or non-peptide library ofcompounds that includes the FKBP-binding domain (FKBP) of the naturalproduct rapamycin or FK506 against the proteins encoded in a genomeusing a protein chip;

b) detecting the binding of a compound to a protein on the chip usingthe anti-V5 antibody together with a fluorescently tagged secondaryantibody;

c) recording the fluorescence pattern of the protein chip on a chipreader;

d) identifying the proteins based on the physical location of thefluorescent spots on the chip; and

e) determining the structure of the lead compound.

In another embodiment, the disclosure provides methods for generating alead compound as a high-affinity ligand of the FKBP isoforms, whereinthe genome is the human genome; and the library of compounds has FormulaI:

or a pharmaceutically acceptable salt or solvate thereof, wherein:

is a single or double bond;

X₁ is O or NR⁶;

Y is —C(O)— or

X₂ is (CH₂)_(m), O, or NR⁶;

Z is

W is O, CH, CH₂, CR⁴, or CR⁵;

L₁ and L₂ are each independently a direct bond, substituted orunsubstituted —(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)O(C₁-C₆)alkyl-, substituted or unsubstituted —(CH₂)_(n)C(O)—,substituted or unsubstituted —(CH₂)_(n)C(O)(C₁-C₆)alkyl-, substituted orunsubstituted —(CH₂)_(n)C(O)O(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)NH(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)S(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₁-C₆)alkyl-, substituted or unsubstituted—(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)O(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)C(O)O(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)NH(C₁-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)S(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkenyl-, substituted or unsubstituted—(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)O(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)C(O)O(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)NH(C₁-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)S(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkynyl-, wherein each alkyl, alkenyl andalkynyl group may be optionally substituted with alkyl, alkoxy, amino,carboxyl, cyano, nitro, or trifluoromethyl;

each m is independently an integer selected from 0, 1, 2, 3, 4, 5, and6;

each n is independently an integer selected from 0, 1, 2, 3, 4, 5, and6;

R¹ is hydrogen, hydroxyl, or OPG, wherein PG is a protecting group, or

R¹ is

wherein

is a resin;

R² is hydrogen, hydroxyl, or alkoxy;

R³ is hydrogen or alkyl;

R⁴ and R⁵ are each independently hydrogen, hydroxy, alkyl, alkoxy, orOPG, wherein PG is a protecting group;

R⁶ is hydrogen or alkyl;

wherein the Effector Domain has Formula II:

wherein:

R⁷, R⁹, R¹¹, R¹³, and R¹⁵ are each independently hydrogen or alkyl;

R⁸, R¹⁰, R¹², and R¹⁴ are each independently hydrogen, halogen, amino,cyano, nitro, trifluoromethyl, substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted perfluoroalkyl, substituted or unsubstituted alkoxy,substituted or unsubstituted alkylamino, substituted or unsubstitutedalkylthio, substituted or unsubstituted aryl, substituted orunsubstituted alkylaryl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted heteroaryl, substituted or unsubstituted heteroalkylaryl,(CH₂)_(n)CN, (CH₂)_(n)CF₃, (CH₂)_(n)C₂F₅, (CH₂)_(n)OR¹⁶,(CH₂)_(n)C(O)R¹⁶, (CH₂)_(n)C(O)OR¹⁶, (CH₂)_(n)OC(O)R¹⁶,(CH₂)_(n)NR¹⁷R¹⁸, (CH₂)_(n)C(O)NR¹⁷R¹⁸, (CH₂)_(n)N¹⁹RC(O)R¹⁶,(CH₂)_(n)N¹⁹RC(O)OR¹⁶, (CH₂)_(n)NR¹⁹C(O)NR¹⁷R¹⁸, (CH₂)_(n)SR¹⁶,(CH₂)_(n)S(O)_(j)NR¹⁷R¹⁸, (CH₂)_(n)N¹⁹RS(O)_(j)R¹⁶, or—(CH₂)_(n)NR¹⁹S(O)_(j)NR¹⁷R¹⁸;

n is an integer selected from 0, 1, 2, 3, 4, 5, and 6;

j is an integer selected from 0, 1, and 2;

R¹⁶, R¹⁷, R¹⁸, and R¹⁹ are each independently hydrogen, halogen, amino,cyano, nitro, trifluoromethyl, alkyl, alkenyl, alkynyl, cycloalkyl,perfluoroalkyl, alkoxy, alkylamino, alkylthio, aryl, alkylaryl,heteroalkyl, heterocycloalkyl, heteroaryl, or heteroalkylaryl, or

R¹⁶ and R¹⁹ are as described above, and R¹⁷ and R¹⁸, together with the Natom to which they are attached, form a substituted or unsubstituted 5-,6-, or 7-membered heterocycloalkyl or a substituted or unsubstituted5-membered heteroaryl,

wherein each of the above groups listed for R⁸, R¹⁰, R¹², and R¹⁴ may beoptionally independently substituted with 1 to 3 groups selected fromhalogen, amino, cyano, nitro, trifluoromethyl, alkyl, alkenyl, alkynyl,cycloalkyl, perfluoroalkyl, alkoxy, alkylamino, alkylthio, aryl,alkylaryl, heteroalkyl, heterocycloalkyl, heteroaryl, heteroalkylaryl,(CH₂)_(n)CN, (CH₂)_(n)CF₃, (CH₂)_(n)C₂F₅, (CH₂)_(n)OR¹⁶,(CH₂)_(n)C(O)R¹⁶, (CH₂)_(n)C(O)OR¹⁶, (CH₂)_(n)OC(O)R¹⁶,(CH₂)_(n)NR¹⁷R¹⁸, (CH₂)_(n)C(O)NR¹⁷R¹⁸, (CH₂)_(n)N¹⁹RC(O)R¹⁶,(CH₂)_(n)N¹⁹RC(O)OR¹⁶, (CH₂)_(n)NR¹⁹C(O)NR¹⁷R¹⁸, (CH₂)_(n)SR¹⁶,(CH₂)_(n)S(O)_(j)NR¹⁷R¹⁸, (CH₂)_(n)N¹⁹RS(O)_(j)R¹⁶, and—(CH₂)_(n)NR¹⁹S(O)_(j)NR¹⁷R¹⁸.

In another embodiment, the disclosure provides methods for generating alead compound as a high-affinity ligand of the FKBP isoforms, whereinthe compound of Formula I:

X is O or NR⁶;

L₁ and L₂ are each independently —(C₁-C₆)alkyl-,—(CH₂)_(n)O(C₁-C₆)alkyl-, —(CH₂)_(n)C(O)(C₁-C₆)alkyl-,—(CH₂)_(n)C(O)O(C₁-C₆)alkyl-, —(CH₂)_(n)NH(C₁-C₆)alkyl-,—(CH₂)_(n)C(O)NH(C₁-C₆)alkyl-, —(CH₂)_(n)S(C₁-C₆)alkyl-,—(CH₂)_(n)C(O)(CH₂)_(n)S(C₁-C₆)alkyl-, —(C₂-C₆)alkenyl-,—(CH₂)_(n)O(C₂-C₆)alkenyl-, —(CH₂)_(n)C(O)(C₂-C₆)alkenyl-,—(CH₂)_(n)C(O)O(C₂-C₆)alkenyl-, —(CH₂)_(n)NH(C₁-C₆)alkenyl-,—(CH₂)_(n)C(O)NH(C₂-C₆)alkenyl-, —(CH₂)_(n)S(C₂-C₆)alkenyl-,—(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkenyl-, wherein each alkyl and alkenylgroup may be substituted with alkyl, alkoxy, or carboxyl;

n is an integer selected from 0, 1, 2, 3, 4, 5, and 6;

R¹ is hydrogen, hydroxyl, or OPG, wherein PG is a silyl protectinggroup, or

R¹ is

wherein

is a resin;

R² is hydroxyl or alkoxy;

R³ is hydrogen or alkyl;

R⁴ and R⁵ are each independently hydrogen, alkyl, alkoxy, or OPG,wherein PG is a silyl protecting group;

R⁶ is hydrogen;

R⁷, R⁹, R¹¹, R¹³, and R¹⁵ are each independently hydrogen or CH₃;

R⁸, R¹⁰, R¹², and R¹⁴ are each independently substituted orunsubstituted cyclopropyl, substituted or unsubstituted cyclobutyl,substituted or unsubstituted cyclopentyl, substituted or unsubstitutedcyclohexyl, substituted or unsubstituted cycloheptyl, or substituted orunsubstituted cyclooctyl; or

R⁸, R¹⁰, R¹², and R¹⁴ are each independently substituted orunsubstituted tetrahydrofuranyl, substituted or unsubstitutedtetrahydrothiophenyl, substituted or unsubstituted pyrrolidinyl,substituted or unsubstituted 1,3-dioxolanyl, substituted orunsubstituted pyrazolidinyl, substituted or unsubstitutedimidazolidinyl, substituted or unsubstituted 1,4-dioxanyl, substitutedor unsubstituted piperidinyl, substituted or unsubstituted piperazinyl,substituted or unsubstituted morpholinyl, substituted or unsubstitutedthiomorpholinyl, or substituted or unsubstituted 1,4-dithianyl; or

R⁸, R¹⁰, R¹², and R¹⁴ are each independently substituted orunsubstituted phenyl, substituted or unsubstituted benzyl, substitutedor unsubstituted naphthylenyl, or substituted or unsubstituted biphenyl;or

R⁸, R¹⁰, R¹², and R¹⁴ are each independently substituted orunsubstituted furanyl, substituted or unsubstituted thiophenyl,substituted or unsubstituted pyrrolyl, substituted or unsubstitutedpyrazolyl, substituted or unsubstituted imidazolyl, substituted orunsubstituted triazolyl, substituted or unsubstituted isoxazolyl,substituted or unsubstituted oxazolyl, substituted or unsubstitutedthiazolyl, substituted or unsubstituted isothiazolyl, substituted orunsubstituted pyridinyl, substituted or unsubstituted pyridizanyl,substituted or unsubstituted pyrimidinyl, substituted or unsubstitutedtriazinyl, substituted or unsubstituted benzofuranyl, substituted orunsubstituted benzo(b)thiophenyl, substituted or unsubstituted indolyl,substituted or unsubstituted benzimidazolyl, substituted orunsubstituted indazolyl, substituted or unsubstituted benzisoxazolyl,substituted or unsubstituted benzoxazolyl, substituted or unsubstitutedbenzothiazolyl, substituted or unsubstituted quinolinyl, substituted orunsubstituted isoquinolinyl, substituted or unsubstituted quinazolinyl,substituted or unsubstituted quinoxalinyl, or substituted orunsubstituted naphthyridinyl.

In another embodiment, the disclosure provides methods for generating alead compound as a high-affinity ligand of the FKBP isoforms, whereinthe compound of Formula I:

L₁ and L₂ are each independently —(C₁-C₆)alkyl-,—(CH₂)_(n)O(C₁-C₆)alkyl-, —(CH₂)_(n)C(O)(C₁-C₆)alkyl-,—(CH₂)_(n)NH(C₁-C₆)alkyl-, —(CH₂)_(n)C(O)NH(C₁-C₆)alkyl-,—(C₂-C₆)alkenyl-, —(CH₂)_(n)O(C₂-C₆)alkenyl-,—(CH₂)_(n)C(O)(C₂-C₆)alkenyl-, —(CH₂)_(n)NH(C₁-C₆)alkenyl-,—(CH₂)_(n)C(O)NH(C₂-C₆)alkenyl-, wherein each alkyl and alkenyl groupmay be substituted with alkyl, alkoxy, or carboxyl;

n is an integer selected from 0, 1, 2, 3, 4, 5, and 6;

R¹ is hydrogen, hydroxyl or OPG, wherein PG is a tert-butyldimethylsilylprotecting group, or

R¹ is

wherein

is Wang resin;

R⁴ and R⁵ are each independently hydrogen, hydroxy, alkyl, alkoxy, orOPG, wherein PG is a tert-butyldimethylsilyl protecting group;

R⁷, R⁹, R¹¹, R¹³ and R¹⁵ are each independently hydrogen;

R⁸, R¹⁰, R¹², and R¹⁴ are each independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted benzyl, substituted orunsubstituted pyrrolidinyl, substituted or unsubstituted indolyl,(CH₂)_(n)OR⁵, (CH₂)_(n)C(O)NR⁶R⁷, or (CH₂)_(n)SR⁵; and

R¹⁶, R¹⁷, and R¹⁸ are each independently hydrogen or (C₁-C₆)alkyl.

In another embodiment, the disclosure provides methods for generating alead compound as a high-affinity ligand of the FKBP isoforms, whereinthe compound of Formula I:

L₁ and L₂ are each independently —(C₁-C₆)alkyl-, —O(C₁-C₆)alkyl-,—C(O)(C₁-C₆)alkyl-, —(CH₂)_(n)C(O)NH(C₁-C₆)alkyl-, —(C₂-C₆)alkenyl-,—O(C₂-C₆)alkenyl-, —C(O)(C₂-C₆)alkenyl-,—(CH₂)_(n)C(O)NH(C₂-C₆)alkenyl-, wherein each alkyl and alkenyl groupmay be substituted with alkyl, alkoxy, or carboxyl;

n is an integer selected from 0, 1, 2, 3, 4, 5, and 6;

R⁸, R¹⁰, R¹², and R¹⁴ are each independently H, CH₃, CH₂OH, CH₂SH,CH(OH)CH₃, CH₂C(O)NH₂, CH₂CH₂C(O)NH₂, CH₂CH₂SCH₃, CH₂CH(CH₃)₂,CH(CH₃)CH₂CH₃, CH₂C₆C₅,

In another embodiment, the disclosure provides methods for generating alead compound as a high-affinity ligand of the FKBP isoforms, whereinthe compound of Formula I:

L₁ and L₂ are each independently —OCH₂CH₂—, —CH₂C(O)—, —CH₂CH₂C(O)—,—C(O)NHCH₂CH₂, —CH₂CH═CHCH₂—, —OCH₂CH═CHCH₂CH₂—, —OCH₂CH═CHCH₂CH(CO2H)—,—CH₂C(O)NHCH₂CH₂—, or CH₂CH(OCH₃)═C(CH₃)CH₂CH₂; and

R⁸, R¹⁰, R¹², and R¹⁴ are each independently the sidechain of the aminoacid alanine, asparagine, cysteine, glutamine, glycine, isoleucine,leucine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, or valine. In another embodiment, the disclosureprovides pharmaceutical compositions including the compound of Formula Iand a pharmaceutically acceptable vehicle. The present invention isbased on the finding that the FKBP-binding domain (FKBP) of theimmunophilin ligand family of macrolide natural products provides ascaffold for combinatorial peptide and non-peptide libraries and alsoacts as an embedded universal tag for each compound in the library. Inparticular, the FKBP-binding domain (FKBD) of the natural productrapamycin can serve both as a scaffold to present combinatorial peptideand non-peptide libraries and as an embedded universal tag for eachcompound in the library. Such hybrid combinatorial libraries areamenable to proteome-wide screening using protein chips by exploitingthe presence of FKBD, which binds to and can be detected with afluorescently labeled antibody against a tagged FKBP. In addition, FKBDcan also confer stability and cell permeability to the fused ligands,increasing the probability that hits from such hybrid combinatoriallibraries are readily applicable to study the cellular functions of therelevant target proteins. This work has lead to a new structural classof ligands that can be used as probes of protein functions.

The immunophilin ligand family consists of three members, cyclosporine A(CsA), FK506 and rapamycin, all of which are natural products withpotent immunosuppressive or anticancer activities. Unlike otherbioactive small molecules, these natural products have an unprecedentedand extraordinary mode of action-through induction of dimeric ternarycomplexes between two distinct proteins: FKBP and their respectiveprotein targets. They each bind to abundant and small cytosolicimmunophilins, which also possess peptidyl prolyl cis-trans isomeraseactivity and are implicated in protein folding. Thus, CsA binds thecyclophilin (CyP) family of immunophilins; and FK506 and rapamycin bothbind FKBP. The formation of the immunophilin-drug complexes per se doesnot have significant cellular consequences. It is the subsequent bindingof these complexes to their respective target proteins that leads toinhibition of T cell activation or tumor cell growth. In the case of CsAand FK506, the CyP-CsA and FKBP-FK506 complexes bind to and inhibit theenzymatic activity of the protein phosphatase calcineurin. In the caseof rapamycin, the FKBP-rapamycin complex binds to the PI3 kinasehomologue known as the Target of Rapamycin (TOR, also known asFKBP-rapamycin associated protein/FRAP and rapamycin and FKBP12target/RAFT).

The crystal structures of the FKBP-FK506-calcineurin andFKBP-rapamycin-TOR complexes revealed that both FK506 and rapamycin canbe divided into two functional domains, the “FKBP-binding domain” (FKBD)and the “effector” domain, which mediate their interactions withcalcineurin and TOR, respectively (FIG. 1). While there are extensiveprotein-protein interactions between FKBP and calcinerin in theirternary complex, there are far fewer interactions between FKBP and TOR,suggesting that the key role of FKBP in the inhibition of TOR byrapamycin is to bind to FKBD of the drug and present its effector domainto TOR.

A comparison of the structures of FK506 and rapamycin reveal that theyshare a nearly identical FKBD but each possesses a distinct effectordomain. By swapping the effector domain of FK506 with that of rapamycin,it is possible to change the target from calcineurin to TOR, which bearsno sequence, functional or structural similarities to each other. Inaddition, other proteins may be targeted by grafting new structures ontothe FKBD of FK506 and rapamycin. Thus, the generation of new compoundswith new target specificity may be achieved by grafting a sufficientlylarge combinatorial library onto FKBD in conjunction with proteome-widescreens through which each compound in the library is tested againstevery protein in the human proteome. In addition, Rapamycin and FK506are immunosuppressant macrocyclic drugs that are used to preventimmunorejection in organ transplantation, especially in kidneytransplants. Rapamycin was first discovered as a product of thebacterium Streptomyces hygroscopicus in a soil sample from EasterIsland. Rapamycin was originally developed as an antifungal agent butlater was found to have both potent immunosuppressive andantiproliferative properties and may be useful in the treatment ofcertain cancers. FK506 is also an immunosuppressive drug that is mainlyused after allogeneic organ transplant to reduce the activity of thepatient's immune system and lower the risk of organ rejection. It isalso used in a topical preparation in the treatment of atopic dermatitis(eczema), severe refractory uveitis after bone marrow transplants,exacerbations of minimal change disease, and the skin conditionvitiligo. FK506 is a 23-membered macrolide lactone discovered in 1984from the fermentation broth of a Japanese soil sample that contained thebacteria Streptomyces tsukubaensis. It reduces interleukin-2 (IL-2)production by T-cells.

Thus, in some embodiments, the compounds of Formula I may be useful asan immunosuppressive agent. These compounds may be delivered to arecipient, prior to, simultaneous with, and/or after transplantation. Inparticular, the compound of Formula I may be administered to cause animmunosuppressive effect in a subject, such that the transplanted cellsare not rejected by that subject's immune system. Typically, theimmunosuppressive agent of Formula I may be administered continuouslythrough-out the transplant treatment typically over a period of days orweeks; for example, treatment may range from about 2 to about 20 days ata dosage range of about 5 to 40 mg per kilogram of body weight per day.The compound of Formula I may be administered by a variety of means,including parenteral, subcutaneous, intrapulmonary, oral, intranasaladministration and the like. Preferably, dosing is given by oraladministration.

Rapamycin and its derivatives including the compound of Formula I, arepromising therapeutic agents with both immunosuppressant and anti-tumorproperties. These actions are mediated through the specific inhibitionof the mTOR protein kinase. mTOR serves as part of an evolutionarilyconserved signaling pathway that controls the cell cycle in response tochanging nutrient levels. The mTOR signaling network contains a numberof tumor suppressor genes including PTEN, LKB1, TSC1, and TSC2, and anumber of proto-oncogenes including PI3K, Akt, and eIF4E, and mTORsignaling is constitutively activated in many tumor types. Theseobservations point to mTOR as an ideal target for anti-cancer agentsincluding rapamycin and the compounds of Formula I. Rapamycinderivatives including the compounds of Formula I, may have efficacy asanti-tumor agents both alone, and when combined with other modes oftherapy. These compounds inhibit tumor growth by halting tumor cellproliferation, inducing tumor cell apoptosis, and suppressing tumorangiogenesis. The immunosuppressant actions result from the inhibitionof T and B cell proliferation through the same mechanisms that rapamycinblocks cancer cell proliferation. Thus, in addition toimmunosuppression, rapamycin derivatives including the compounds ofFormula I may act as anti-cancer agents.

The compounds of Formula I may be useful in the treatment ofhyperproliferative disorders, for example cancer, including melanoma andother cancers of the skin, breast cancer, bladder cancer, colon cancer,glioma, glioblastoma, lung cancer, hepatocellular cancer, gastriccancer, melanoma, thyroid cancer, endometrial cancer, renal cancer,cervical cancer, pancreatic cancer, esophageal cancer, prostate cancer,brain cancer, and ovarian cancer, neurodegeneration, cardiachypertrophy, pain, migraine, neurotraumatic diseases, stroke, diabetes,hepatomegaly, cardiovascular disease, Alzheimer's disease, cysticfibrosis, autoimmune diseases, atherosclerosis, restenosis, psoriasis,allergis disorders, inflammation, neurological disorders,hormone-related diseases, conditions associated with organtransplantation, immunodeficiency disorders, destructive bone disorders,hyperproliferative disorders, infectious diseases, conditions associatedwith cell death, thrombin-induced platelet aggregation, chronicmyelogenous leukaemia (CML), liver disease, pathologic immune conditionsinvolving T cell activation, and CNS disorders. The compounds of FormulaI may also be useful for treating a condition by modulation of mTORactivity by administering to a human or animal subject in need of suchtreatment an effective amount of the compound.

The design and synthesis of combinatorial libraries fused to the FKBPbinding domain of rapamycin is premised on the finding that replacementof the effector domain of rapamycin by a tetrapeptide, providesrapamycin hybrid compounds that retain most of the high affinityFKBP-binding activity of rapamycin.

Previous synthesis of the FKBD from rapamycin involved the initialcleavage of the inherently labile ester bond at C-34 of rapamycin, whichnecessitated the reassembly of the two resulting pieces of FKBD.Alternatively, the disclosure provides a simpler and more efficientmethod to prepare the desired FKBP from rapamycin while keeping theentire FKBD intact throughout the process.

As shown below in Scheme 1, degradation of rapamycin (1) and reassemblyof two fragments (4) and (5) provide the core structure (6) that isrequired for FKBP binding.

Commercially available rapamycin (1) is first subjected to silylprotection of the C-40, C-28, and C-10 hydroxyl groups usingtert-butyldimethylsilyl triflate ((a) 3 equivalents of TBDSOTf orTBSOTf, 2,6 lutidine, triethylamine (Et₃N), and dichloromethane (CH₂Cl₂)at 0° C.). Other suitable hydroxyl protecting groups include but are notlimited to methoxymethyl ether (MOM), tetrahydropyranyl ether (THP),tert-butyl ether (t-Bu), allyl ether, benzyl ether,tert-butyldiphenylsilyl ether (TBDPS), acetic acid ester, pivalic acidester, benzoic acid ester, and the like.

The triprotected rapamycin is then fragmented using exhaustiveozonolysis via a known optimized protocol ((b) 0₃, −78° C.,CH₂Cl₂/CH₃OH, then (CH₃)₂S) to afford the pipecolate fragment (2) andcyclohexane-containing enone (3).

Pipecolic acid (4) is readily derived from the pipecolate fragment (2)by installing the methylene group on C-17 (rapamycin numbering) using aWittig reaction ((f) (Ph₃P⁺Me)I⁻¹, NaH, THF) and saponification of theester (at C-31) ((g) aqueous LiOH, THF) to provide the desiredpipecolate (4) for eventual coupling. Fragment (3) is further degradedby another exhaustive ozonolysis ((b) 0₃, −78° C., CH₂Cl₂/CH₃OH, then(CH₃)₂S) to yield an intermediate aldehyde, which upon stereoselectiveStrecker synthesis ((h) CO/(PPh₃)₂PdBr₂/LiBr/H₂SO₄, DMF; then enzymehydrolysis) yields the required amino acid (5). Finally, preparation ofN-hydrosuccinimide active ester of pipecolate (4) ((i) 4 plus NHS, DCC,DMAP, CH₂Cl₂), and subsequent condensation with amino acid (5) ((j)Piperidine, DMF) provides the FKBD of rapamycin (6), which is ready forpeptide coupling at the proximal pipecolic acid end and a ring-closingmetathesis reaction at the distal alkenyl end.

Alternatively, as shown below in Scheme 2, the synthesis of the FKBDfrom rapamycin may be accomplished as follows.

Treatment of rapamycin (1) with tert-butyldimethylsilyl triflate andtriethyl amine ((a)TBSOTf, Et₃N, CH₂Cl₂, room temperature, 100%) affordsthe fully protected epimers (2) in quantitative yield. According to anearlier report, protection of the hydroxyl at the hemiketal center(C-10) of rapamycin is not required following the ozonolysis step.However, the corresponding C-10 unprotected FKBD fragment may be cleavedbetween C-9 and C-10 when subjected to Baeyer-Villiger oxidation. Thus,protection of the C-10 hydroxyl is required in this scheme. Therapamycin derivative (2) is subjected to ozonolysis and subsequentlytreated with hydrogen peroxide ((b) O₃, CH₂Cl₂ at −68° C., and 35% H₂O₂)to give an intermediate containing a carboxylic acid at one end and aperoxyhemiacetal or aldehyde at the other end of the FKBD fragment.After a brief flash-column chromatography over silica gel, the crudeproduct was directly subjected to Wittig olefination ((c) CH₃PPh3Br,t-BuOK, THF, 0° C.) to acid (3) in 54% combined yield. Deprotection ofthe silyl protecting groups ((d) trifluoroacetic acid and H₂O at 0° C.)provides the product FKBD (4) in 96% yield. It is noteworthy thatneither tetrabutylammonium fluoride (TBAF) nor HF-pyridine were able toaccomplish a clean deprotection of the two silyl groups. Thus, the FKBDmay be prepared from rapamycin in four steps with 52% overall yield.

The intermediates after ozonolysis and the Baeyer-Villiger reaction werenot capable of being analyzed due in part to the complexity of thereaction products. The complexity was further compounded by thepropensity of the FKBD to exhibit rotamerism and in this case we werealso dealing with a pair of epimers. As shown below in Scheme 3, themost likely intermediates in the course of these two tandem steps(ozonolysis/Baeyer-Villiger) can be predicted as follows.

Ozonolysis of the C29-C30 double bond gave rise to aldehyde intermediate(5) that could undergo facile enolization to give 6. This rendered 6with an exclusive migratory aptitude in the Baeyer-Villigerrearrangement to give ester 7. Hydrolysis of 7 led to the formation ofthe carboxylic group in 3. Concurrently, ozonolysis of C17-C18 doublebond yielded the ketone intermediate 8. Baeyer-Villiger oxidation of 8led to the formation, possibly via ester 9, of peroxyhemiacetal (10)that, upon hydrolysis, gave the corresponding aldehyde 11, which isready to undergo Wittig reaction to form the terminal olefin in 3. Aninteresting feature of this procedure is that the same set of reactionsoccurred concomitantly at both ends of FKBD, but led to the formation ofdistinct functional groups-a carboxylic acid and an olefinic group. Thissimultaneous chemical transformation of two groups significantly reducedthe number of steps required to prepare FKBD, hence the high overallyield.

In addition to serving our purpose for the synthesis of FKBD-containinghybrid combinatorial libraries, high-affinity ligands of FKBP isoformsmay find use as probes for a number of biological processes, as FKBPthemselves have been implicated in the regulation of neuronal celldifferentiation, ion channels, and Ras post-translational modificationamong others. The easy access to FKBD could thus allow for the synthesisof potent and possibly iso-form-selective FKBP ligands that are devoidof immunosuppressive and antiproliferative activity of FK506 andrapamycin.

For the methods of making the libraries, there are several differentvariations of achieving the macrocyclization. They include ring-closingmetathesis (Scheme 4, 7, 11), safety catch method (Scheme 6, 10), thioldisplacement (Scheme 14) and amide bond formation (Scheme 15).As shownbelow in Scheme 4, the synthesis of a peptide library using the FKBD ofrapamycin (6), can be prepared using the well-established solid-phasesplit-pool peptide synthesis method.

The library may be anchored to resin via a pre-installed olefinic groupin order to enable the final cyclization step using olefin metathesischemistry. Thus, the known Boc-protected a,y-hydroxyalkenylamine linker(7) may be charged onto commercially available Wang resin using awell-established protocol ((a) 7 then, NaH/THF; add to Wang resin) toprovide (8). After deprotection with trifluoroacetic acid to remove theBoc-protecting group ((b) 15% TFA/CH₂Cl₂), the resin is ready for aseries of amino acid coupling steps using conventional Fmoc-basedsolid-phase peptide synthesis ((c) Fmoc-AA-OH, PyBOP, DIPEA, NMP). Thus,the amino acid couplings may be repeated until a tetrapeptide (9) isbuilt on the resin. The tetrapeptides, together with the added spacer,provides for a ring size that is comparable to that of rapamycin. Forbuilding blocks, the five charged amino acids, Asp, Glu, His, Arg, andLys are omitted to make the resulting tetrapeptides more hydrophobic, asthe effector domains for both FK506 and rapamycin are bound tohydrophobic pockets of calcineurin and TOR. This protocol leads to alibrary of 154 or 50,625 individual peptides. To suit the Fmoc-strategyand to set up for a one-step deprotection in the end, compatibleprotecting groups for side chains of the amino acid building blocks areutilized, i.e., trityl or tert-butyl for Ser, Cys, Thr, and Tyr.

In addition to alpha and beta amino acids, the Effector Domain mayinclude N-methylated and N-alkylated amino acids as well as both D- andL-amino acids. Other useful amino acids include but are not limited topeptoids, peptidomimetic, depsipeptides, and mixtures thereof.

Upon completion of the tetrapeptide synthesis, the precursor of FKBD ofrapamycin, (6), may be coupled to the C-terminal carboxylate group of(9). The cyclization of the peptides (10) may be achieved using ametathesis reaction with a Grubb's second-generation catalyst,accompanied with the release of the cyclic peptides from the resin ((e)(IMesH₂)(PCy₃)(CI₂)Ru═CHPh, CH₂Cl₂). Finally, the products may betreated with trifluoroacetic acid to remove the silyl protection on FKBDof rapamycin and all the amino acid side-chain protecting groupssimultaneously in one step ((b) 15% TFA/CH₂Cl₂). The beads may befiltered and removed and the desired library products are ready for highthroughput screening by simply evaporating the volatiles from thereaction under high vacuum. The composition and integrity of each poolof compounds may be determined using LC-Mass spectrometry.

PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphoniumhexafluorophosphate) is a peptide coupling reagent used in solid phasepeptide synthesis. It is used as a substitute for the BOP reagent, thusavoiding the formation of the carcinogenic side product HMPA.

N,N-Diisopropylethylamine, or Hünig's base, DIPEA or DIEA, is an organiccompound and an amine. It is used in organic chemistry as a base.Because the nitrogen atom is shielded by the two isopropyl groups and anethyl group only a proton is small enough to easily fit. Similar to2,2,6,6-tetramethylpiperidine, this compound is a good base but a poornucleophile, which makes it a useful organic reagent.

Wang resin (4-benzyloxybenzyl alcohol resin) is the most popular supportfor solid phase organic synthesis (SPOS) using Fmoc chemistry. As astandard support it can be used for the solid phase immobilization ofacids and phenols for SPOS. The ester linkage may be achieved, which hasgood stability to a variety of reaction conditions, but can be readilyremoved with the moderate acid treatment, generally with trifluoroaceticacid. For the immobilization of amines, Wang resin also can be readilyconverted into solid phase equivalents of standard urethane-basedprotecting groups by reaction with phosgene or activated carbonates,such as carbonyl diimidazole or bis(p-nitropnenyl)-carbonate. Othersuitable resins include but are not limited to polystyrene resins suchas aminomethyl polystyrene resin, 2-chrlotrityl chloride resin, DHP HMresin, HMPA-AM resin, Knorr resin, Knorr-2-chlorotrityl resin, MBHAresin, Merrifield resin, oxime resin, PAM resin, Rink amide-AM resin,Rink amide-MBHA resin, Sieber resin, Wang resin, Weinreb AM resin,Boc-Ser-Merrifield resin, and Boc-Gly-Merrifield resin, and the like.

The “one-bead-one-compound” (OBOC) combinatorial library method may beused to synthesize millions of compounds such that each bead displaysonly one compound. Bead libraries are screened, and positive beads areisolated for structure analysis.

The linking group (linker) that joins the substrate to the resin bead isan essential part of solid phase synthesis. The linker is a specializedprotecting group, in that much of the time, the linker will tie up afunctional group, only for it to reappear at the end of the synthesis.The linker must not be affected by the chemistry used to modify orextend the attached compound. The cleavage step should proceed readilyand in a good yield as the best linker allows for attachment andcleavage in quantitative yield.

Grubbs' Catalyst is a transition metal carbene complex named afterRobert H. Grubbs, the chemist who first synthesized it. There are twogenerations of the catalyst, as shown below:

In contrast to other olefin metathesis catalysts, Grubbs' Catalyststolerate other functional groups in the alkene and are compatible with awide range of solvents. For these reasons, Grubbs' Catalysts areextraordinarily versatile.

The First Generation Catalyst is often used in organic synthesis toachieve olefin cross-metathesis, ring-opening metathesis polymerization(ROMP), acyclic diene metathesis polymerization (ADMET), andring-closing metathesis. It is easily synthesized from RuCl₂(PPh₃)₃,phenyldiazomethane, and tricyclohexylphosphine in a one-pot synthesis.Grubbs' Catalyst is a relatively stable compound in air, which makeshandling very easy. The IUPAC name of the 1st Generation Catalyst isbenzylidene bis(tricyclohexylphosphine)-dichlororuthenium. The SecondGeneration Catalyst has the same uses in organic synthesis as the FirstGeneration Catalyst, but has a higher activity. This catalyst is stabletoward moisture and air, thus it is easier to handle in the lab. TheIUPAC name of the Second Generation Catalyst isbenzylidene[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene]dichloro(tricyclohexyl-phosphine)ruthenium.Both generations of the catalyst are commercially available.

Olefin metathesis is a reaction between two molecules containing doublebonds. The groups bonded to the carbon atoms of the double bond areexchanged between molecules, to produce two new molecules containingdouble bonds with swapped groups. Whether a cis isomer or trans isomeris formed in this type of reaction is determined by the orientation themolecules assume when they coordinate to the catalyst, as well as thesteric interactions of the substituents on the double bond of the newlyforming molecule. Other catalysts are effective for this reaction,notably those developed by Richard R. Schrock, i.e., the Schrockcarbene.

In addition to the hybrid combinatorial peptide library described above,there are many more options for both the building blocks of the libraryand the synthetic routes. For example, instead of naturally occurringalpha L-amino acids, beta-amino acids, D-amino acids, p-amino acids,N-methyl amino acids (as seen in CsA) or peptoids, and non-amino acidsmay be used in versions of the hybrid libraries. The lengths of thepeptides or other building blocks can also be decreased or increased aswell. Linkages other than peptide and olefins may also be used toconnect the library to FKBD of rapamycin. These variations can give riseto significantly greater diversity and biochemical properties of thecombinatorial fusion libraries.

Aside from the standard amino acids, there are many other non-standardamino acids that may be used in the disclosed methods and compounds. Forexample, carnitine, hydroxyproline, selenomethionine, hydroxyproline,lanthionine, 2-aminoisobutyric acid, dehydroalanine, and theneurotransmitter gamma-aminobutyric acid (GABA). Nonstandard amino acidsoften occur as intermediates in the metabolic pathways for standardamino acids—for example, ornithine and citrulline occur in the ureacycle, part of amino acid catabolism. A rare exception to the dominanceof α-amino acids in biology is the β-amino acid beta alanineβ-aminopropanoic acid), which is used in plants and microorganisms inthe synthesis of pantothenic acid (vitamin B₅), a component of coenzymeA. Other α-amino acids and β-amino acids are contemplated to be withinthe scope of the invention.

To facilitate the decoding of the hits, two orthogonallysplit-and-pooled libraries with one kept split at the 1^(st) and 3^(rd)and the other at 2^(nd) and 4^(th) coupling steps, respectively may beused (FIG. 2). This provides two partially “decodable” libraries, eachconsisting of 15×15 or 225 individual pools. By screening the firstlibrary, the identities of the 1^(st) and 3^(rd) residues of the hitsmay be known as they remained in different pools. By screening theorthogonally split-and-pooled library, the amino acid identities of the2^(nd) and the 4^(th) residues of the hits may also be known. Thecombined information of the two orthogonally split-and-pooled librariesprovides for the optimal sequences of the hits. It is possible that morethan one residue may show up at a given position, which can be furtherdetermined by synthesizing and testing all possible combinations ofindividual cyclic peptides.

A feature of the soluble libraries is that they are made in a partiallysplit-pooled manner (see FIG. 2). Two sets of each library may be madethat are orthogonally split and pooled. In the end, each pool willcontain a mixture with certain defined positions. By screening the twoorthogonal pools simultaneously, it will be possible to decode theresidues at each and every position, allowing for the decoding of thehits.

The orthogonally split-and-pooled library pairs can be screened usingmost other screening platforms, including cell-based screens orprotein-target based screens in addition to the protein chip. In fact,if there is a predetermined pathway (such as hedgehog, Wnt, Myc etc) ortarget (Bcr-Abl, VEGFR, Her2 etc), the cell-, pathway- or target-basedscreens may be used instead of the more comprehensive whole proteomechip screens.

For the libraries retained on beads, they may be made in a split-poolstrategy so that each bead will contain a single homogeneous compound(so-called one-bead-one-compound or OBOC strategy). These solid-phaselibraries will be screened using the beads directly. This type ofsolid-phase libraries is suitable for screening against selected singleprotein target only. In one format, the target protein may be labeledwith a fluorescence marker. The labeled protein may be incubated withthe solid-phase libraries (with 200,000 to over 1 million individualcompounds on individual beads per library) with the fluorescentlylabeled target protein in the absence and presence of recombinant FKBP.The positive hits may be identified by the fluorescent intensity of thebeads. If a bead contains a positive hit, it is expected that it willbind to the fluorescently labeled target protein. Those beads will thenbe selected using either a micropipet or by flow cytometry. Theidentities of the hits may also be determined using mass spectrometry.As a follow-up, the same compounds may be made individually, releasedinto solution and tested in complementary assays.

Human protein chips may be made from glass slides of 3.5 cm by 7.5 cm indimension. Each chip can be screened in a chamber containing 2-3 mL ofbuffer. To detect the hits that form complexes with FKBP and anotherprotein present on a chip, a recombinant FKBP containing a C-terminal V5peptide epitope tag may be generated against which there are highlyspecific antibodies. The detection is rendered possible by using anti-V5antibody together with a fluorescently tagged secondary antibody (FIG.3). Thus, upon incubation of the library and V5-tagged FKBP, thoseproteins that are bound to V5-FKBP may be detected through the presenceof fluorescence at the locations where the proteins are placed in thearray.

The two orthogonally split-and-pooled libraries may be screened usingone pool per chip. Thus, a total of 450 pools of hybrid compounds can bescreened using 450 chips. Each compound may be present at a finalconcentration of about 1 μM. The total binding capacity of each pool of225 compounds for FKBP is 225 μM. To ensure that all compounds are boundby V5-FKBP protein, V5-FKBP at a final concentration of 500 μM is used,which is easily achievable due to its high aqueous solubility. Uponincubation of V5-FKBP and a pool of the compounds with the proteins onprotein chips, the chips may be washed three times with the bindingbuffer, followed by addition of anti-V5 antibody and afluorescein-tagged secondary antibody. The chips may be further washedbefore its fluorescence pattern is recorded in a chip reader. Theidentities of the proteins may be retrieved based on the physicallocations of fluorescent spots on the chip. As FKBP is known to bind asmall number of proteins itself, a parallel screen in the absence ofadded library may also be conducted, which may serve as a negativecontrol. The fluorescence intensity of each protein spot may bequantitated and normalized against that from the V5-FKBP control. Thoseproteins whose fluorescence intensity becomes significantly higher inthe presence of added hybrid cyclic peptide may be considered to behits.

It is interesting to note that by screening the two libraries of 50,625individual compounds against 450 human protein chips that contain 17,000human proteins on each chip, an equivalent of a total of 2×50,625×17,000or 1,721,250,000 individual binding assays may be accomplished.

For the hits that are bound to a given protein or a set of proteins, thecorresponding tetrapeptide sequences based on the specific pools towhich the hits belong may be decoded. From the two orthogonallysplit-and-pooled libraries, the optimal sequence or sequences of thefour residues against a given protein target may be determined.Accordingly, all possible hybrid ligands may be synthesized and testedindividually using the chip assay. Upon validation on protein chips, apull-down assay with GST-FKBP and the putative recombinant targetprotein in the absence and presence of the hybrid ligands may be used.In the event that the putative target proteins are already known to playa role in a given cellular process, be it cell cycle progression,transcription, translation or apoptosis, the newly identified ligandsmay be subject to the appropriate cell-based assays to determine if theyhave the expected effects on the corresponding cellular processes.

The generation of orthogonally split-and-pooled libraries of hybridcyclic peptides fused to FKBD enables the proteome-wide screening ofhuman protein chips and facilitates the identification of hits that canbe rapidly resynthesized and validated. The combination of the librarieswith the protein chip screening platform renders it possible for thefirst time to perform large-scale screens of combinatorial librariesagainst the entire proteome to identify the small molecule hits and theprotein targets simultaneously. Moreover, unlike compounds fromconventional peptide libraries, the hits from the hybrid cyclic peptideand non-peptide libraries may be endowed with greater stability and cellpermeability due to the presence of FKBD, which may make the hitsreadily applicable as probes of the cellular function of the putativetargets. This approach may lead to a new paradigm for decoding thefunction of proteins in the human proteome, i.e., from proteins tobinding ligands to cellular functions. There are also unlimitedpossibilities beyond the peptides that can be fused to FKBD as well asthe cyclophilin-binding domain of CsA to give rise to even greaterchemical diversity to cover more proteins in the human proteome.

The disclosure also provides methods for administering to a subject atherapeutically effective dose of a pharmaceutical compositioncontaining the compounds of the present invention and a pharmaceuticallyacceptable carrier. “Administering” the pharmaceutical composition ofthe present invention may be accomplished by any means known to theskilled artisan.

The pharmaceutical compositions may be prepared and administered in doseunits. Solid dose units are tablets, capsules and suppositories. Fortreatment of a patient, depending on activity of the compound, manner ofadministration, nature and severity of the disorder, age and body weightof the patient, different daily doses are necessary. Under certaincircumstances, however, higher or lower daily doses may be appropriate.The administration of the daily dose can be carried out both by singleadministration in the form of an individual dose unit or else severalsmaller dose units and also by multiple administration of subdivideddoses at specific intervals.

The pharmaceutical compositions according to the invention are ingeneral administered topically, orally, intravenously, or by anotherparenteral route, or as implants, or even rectal use is possible inprinciple. Suitable solid or liquid pharmaceutical preparation formsare, for example, granules, powders, tablets, coated tablets,(micro)capsules, suppositories, syrups, emulsions, suspensions, creams,aerosols, drops or injectable solution in ampule form and alsopreparations with protracted release of active compounds, in whosepreparation excipients and additives and/or auxiliaries such asdisintegrants, binders, coating agents, swelling agents, lubricants,flavorings, sweeteners or solubilizers are customarily used as describedabove. The pharmaceutical compositions are suitable for use in a varietyof drug delivery systems. For a brief review of present methods for drugdelivery, see Langer, Science, 249:1527-1533, 1990, which isincorporated herein by reference.

For delivery of the compounds, the formulations may be prepared bycontacting the compounds uniformly and intimately with liquid carriersor finely divided solid carriers or both, and then, if necessary,shaping the product into the desired formulation. The carrier may be aparenteral carrier, or a solution that is isotonic with the blood of therecipient. Examples of such carrier vehicles include water, saline,Ringer's solution, dextrose solution, and 5% human serum albumin.Nonaqueous vehicles such as fixed oils and ethyl oleate are also usefulherein, as well as liposomes. Generally, the carrier can contain minoramounts of additives such as substances that enhance isotonicity andchemical stability, e.g., buffers and preservatives, as well as lowmolecular weight (less than about 10 residues) polypeptides, proteins,amino acids, carbohydrates including glucose or dextrans, chelatingagents such as EDTA, or other excipients.

The composition described herein may be suitably administered bysustained release systems. Suitable examples of sustained releasecompositions include semipermeable polymer matrices in the form ofshaped articles, e.g., films, microcapsules, or microspheres. Sustainedrelease matrices include, for example, polyactides (U.S. Pat. No.3,773,919), copolymers of L-glutamic acid and .gamma.-ethyl-L-glutamate(Sidman et al., Biopolymers 22:547-556, 1983), orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988). Sustained releasecompositions also include one or more liposomally entrapped compounds offormula I. Such compositions are prepared by methods known per se, e.g.,as taught by Epstein et al. Proc. Natl. Acad. Sci. USA 82:3688-3692,1985. Ordinarily, the liposomes are of the small (200-800 .ANG.)unilamellar type in which the lipid content is greater than about 30 mol% cholesterol, the selected proportion being adjusted for the optimaltherapy.

The pharmaceutical compositions according to the invention may beadministered locally or systemically. By “therapeutically effectivedose” is meant the quantity of a compound according to the inventionnecessary to prevent, to cure or at least partially arrest the symptomsof the disorder and its complications. Amounts effective for this usewill, of course, depend on the severity of the disease and the weightand general state of the patient. Typically, dosages used in vitro mayprovide useful guidance in the amounts useful for in situ administrationof the pharmaceutical composition, and animal models may be used todetermine effective dosages for treatment of particular disorders.Various considerations are described, e.g., in Gilman et al., eds.,Goodman And Gilman's: The Pharmacological Bases of Therapeutics, 8thed., Pergamon Press, 1990; and Remington's Pharmaceutical Sciences, 17thed., Mack Publishing Co., Easton, Pa., 1990; each of which is hereinincorporated by reference.

The following examples are intended to illustrate but not limit theinvention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may alternatively be used.

EXAMPLES

This invention is illustrated by the following exemplar embodiments,which are not to be construed in any way as imposing limitations on thescope thereof. On the contrary, various other embodiments,modifications, and equivalents thereof, which, after reading thedescription herein, may suggest themselves to those skilled in the art,may be made without departing from the spirit or scope of the presentinvention. The following examples are intended to illustrate but notlimit the invention.

Example 1 Synthesis of 2 in Scheme 2

To a solution of rapamycin (999 mg, 1.09 mmol) and anhydroustriethylamine (Et₃N) (0.6 mL, 4.45 mmol) in anhydrous dichloromethane(CH₂Cl₂) (3 mL) is added tert-butyldimethylsilyl triflate (TBSOTf) (0.85mL, 3.79 mmol). The solution is stirred for 1 hour at room temperatureand quenched with water. The resulting mixture is diluted with ethylacetate (EtOAc) (120 mL), washed with saturated sodium bicarbonate(NaHCO₃) and brine, and dried and concentrated in vacuo. The cruderesidue is subjected to silica gel column chromatography (Hexanes/EtOAc,8:1) to afford 2 (1.37 g, 100*) as a yellow solid: NMR (400 MHz, CDCl₃):δ 6.39-5.82 (m, 4 H), 5.52-4.91 (m, 4 H), 4.45-3.98 (m, 2 H), 3.83 (d,7-4.0 Hz, 1 H), 3.40 (s, 3 H), 3.27 (s, 3 H). 3.10 (s. 3 H), 2.89-2.83(m, 1 H), 2.66-2.40 (m, 3 H), 2.23-2,19 (m, 3 H), 1.70 (s, 3 H), 0.17-0(m, 18 H); ¹³C NMR (100 MHz, CDCl₃): δ 210.3, 206.6, 197.9, 169.5,167.0, 139.3, 139.0, 136.6, 132.0, 130.8, 127.4, 126.7, 126.4, 101.7,86.6, 84.3, 83.2, 78.0, 75.7, 74.3, 67.7, 58.1, 57.8. 56.1, 51.4. 45.8,44.1. 41.4, 40.8, 40.6, 40.2, 38.2, 36.8, 35.7, 34.0. 33,9, 33.0, 32.0,30.1, 27.1, 26.6, 26.3, 26.0, 25.9, 25.8, 25.7, 24.9, 22.0, 21.0, 19.3,18.2, 18.1, 15.7, 15.5, 14.0, 13.9, 11.0, −2.5, −3.2, −4.5, −4.6, −4.7,−4.9; HR-ES1MS calcd for C₆₉H₁₂₁O₁₃NSi₃Na [M+Na]* 1278.8043, found1278.8049.

Example 2 Synthesis of 3 in Scheme 2

Toa solution the silyl derivative 2 (1.20 g, 0.95 mmol) in CH₂Cl₂ (15mL) at −68° C. is bubbled ozone (0₃) until the blue color persisted. 35%hydrogen peroxide (H₂0₂) (15 mL) is added and the stirring is continuedfor another 14 hours at room temperature. The solution is diluted withEtOAc (120 mL), washed with brine, dried and concentrated to afford anoil that is purified using silica gel chromatography (Hexanes/EtOAc,4:1, then CH₂Cl₂—MeOH, 20:1) to provide an epimeric mixture (710 mg) asa white solid. The epimeric mixture from the ozonolysis reaction statedabove (210 mg, 0.26 mmol) is dissolved in anhydrous THF (2 mL), andadded to a freshly prepared Wittig reagent at 0° C., which in turn wasprepared from CH₃PPh₃Br (560 mg, 1.57 mmol) and tert-BuOK (140 mg, 1.25mmol) in anhydrous THF (5 mL). After stirring for 10 minutes, thereaction mixture is quenched with 5% HCl. The resulting mixture isdiluted with EtOAc (60 mL), washed with brine, dried and concentrated.The crude residue is purified by silica gel chromatography(toluene-EtOAc. 4:1, then toluene-EtOAc-AcOH, 4:1:0.025) to provide 3(124 mg, 54%) as a white solid: ¹H NMR (400 MHz, CDCl₃): 5.84-5.68 (m, 1H). 5.29-4.89 (m. 4 H). 4.42-4.29 (m, 1 H). 4.06-3.85 (m, 1 H), 3.40 (s.3 H), 2.93-2.48 (m, 4 H). 2.34-2.13 (m. 4 H), 0.87 (s, 9 H), 0.19-0.02(m, 12 H); ¹³C NMR (100 MHz, CDCl₃): δ 197.7, 197.4, 176.0, 175.4,169.7, 169.2, 167.2, 166.4, 135.3, 134.3, 117.2, 116.6, 102.0, 101.6,84.5, 84.4, 75.7, 75.2, 74.6, 70.5, 70.3, 58.1, 58.0, 56.6, 56.5, 51.8,44.4, 40.3, 38.9, 38.6, 36.5, 36.4, 36.2, 36.0, 35.1, 34.7, 33.9, 33.34,33.25, 33.1, 31.4, 30.9, 30.6, 29.7, 29.4, 27.6, 27.3, 26.8, 26.76,26.6, 26.3, 25.9, 24.8, 24.4, 22.8, 21.3, 20.7, 19.4, 18.2, 15.82,15.78, 15.3, 14.9, −2.5, −2.7, −2.9, −3.0, −4.5, −4.7: HR-ES1MS calcdfor C₄₂H₇₅0₁₀NSi₂Na [M+Na]*832.4827, found 832.4833.

Example 3 Synthesis of 4 in Scheme 2

To a mixture of trifluoroacetic acid (CF₃C0₂H) (0.8 mL) and H₂0 (0.2 mL)at 0° C. is added olefin 3 (32 mg, 0.040 mmol). After stirring for 2hours, the mixture is concentrated in vacuo and purified by silica gelchromatography (CH₂Cl₂—MeOH—AcOH. 20:1:0.2) to provide 4 (22 mg, 96%) asa white solid: ¹HNMR (400 MHz, CDCl₃): 5.82-5.68 (m, 1 H), 5.27-4.90 (m.4 H), 4.45-4.32 (m, 1 H), 4.02-3.85 (m, 1 H), 3.40 (s, 3 H). 2.99-2.94(m, 1 H), 2.65-2.51 (m, 2 H): ¹³C NMR (100 MHz, CDCl₃): δ 197.8, 194.8,175.1, 175.0, 171.0, 170.0, 169.8, 169.3, 169.3, 167.6, 166.8, 165.7,135.1, 134.8, 134.3, 117.4, 117.1, 116.7, 99.3, 98.5, 97.7, 84.4, 75.7,75.2, 75.0, 73.9, 70.2, 70.1, 56.5, 56.4, 51.5, 44.5, 43.1, 41.5, 41.4,40.2, 39.1, 38.9, 35.1, 35.0, 34.4, 34.3, 34.1, 33.4, 33.2, 31.3, 30.8,30.6, 29.7, 27.5, 27.1, 26.6, 26.4, 26.3, 25.0, 24.6, 24.4, 21.1, 20.9,20,8, 16.7, 16.1, 15.9, 15.7, 15.3, 15.1, 15.0; HR-ESIMS calcd forC₃₀H⁴⁷0₁₀NNa [M+Na]*604.3098, found 604.3093.

Example 4 Synthesis of Macrocycles Containing Mimic of FKBD

The synthesis of novel mimic FKBD fragments one for use in RCMmacrocyclization strategy and one for macrolactamization strategy, isshown below in Scheme 5.

3′-Hydroxy-4′-methoxy acetophenone was dissolved in pyridine andoxidized with selenium dioxide at 125° C. to obtain the desiredglyoxalate. This compound is then coupled with ethyl pipecolate usingEDCI, HOAt, DMAP, DIPEA in to obtain the key intermediate. Theintermediate compound may be modified into either the RCM substrate orsubstrate for macrolactamization.

Macrolactamization Substrate

The key intermediate was reacted with -butyl-2-bromoethylcarbamate inpresence of potassium carbonate and potassium iodide in DMF followed bybase hydrolysis to obtain the desired substrate for solid phase couplingwith D-HomoPhe terminated peptide sequence.

RCM Substrate

The key intermediate was reacted with allyl bromide in presence ofpotassium carbonate in acetone at room temperature followed by basehydrolysis to obtain the desired substrate for solid phase coupling withD-HomoPhe terminated peptide sequence.

Example 5 Macrocycle Macrolactamization Strategy Using Safety CatchLinker

Two different tetrapeptides—Leu-Ala-Val-Gly-D-HomoPhe andAla-Tyr(tBu)-N-MeLeu-Gly-D-HomoPhe were synthesized using standard solidphase coupling protocols. The mimic FKBD substrate was coupled to theterminal D-homophenylalanine followed by activation with ICH₂CN inabsence of light and subsequent removal of all Boc protecting groupsusing 50-80 TFA in DCM. This was followed by the macrolactamization with20% DIPEA in THF and cyclitive release of the desired mimic rapafucin in20-28% yield after purification. Synthesis of one of the peptidesequences is shown in Scheme 6.

Example 6 Macrocycle Ring-Closing Metathesis Strategy

The below linear precursor, with terminal double bonds, from cleavage ofproduct from chlorotrityl chloride resin was subjected to RCM usingGrubb's second generation catalyst in solution phase using microwaveconditions 120° C. for 30 min in DCE as well as traditional hotplate(50° C., overnight) to obtain the desired cyclized product in 70% yieldas shown in Scheme 7.

Example 7 Preparation of Natural FKBD from Rapamycin and Synthesis ofMacrocycles

Treatment of Rapamycin with TBSOTf and Et₃N in DCM offered a fullyprotected silylate, which was applied to ozonolysis and Baeyer-Villigeroxidation to provide a mixture of FKBD fragments as shown in Scheme 8.

Example 8 Preparation of a Tetrapeptide. (Leu-Val-Ala-Gly)

The mixture of FKBD fragments was coupled with under the condition ofHBTU and DIPEA, and the R group was then treated with oxone to result ina carboxyl acid group, followed by coupling with N-Boc-ethylenediamineat 0° C. Deprotection of all the TBS groups with TBAF at 0° C. andsubsequent activation with ICH₃CN in darkness to provide an orangeresin, which was then subjected to 50% TFA in DCM at 0° C. to give afree amino group. Treatment the ring-closed precursor above with 20%DIPEA in THF afforded the desired rapafucin in 16% overall yield asshown in Scheme 9 and 10.

Example 9 Macrocyclization Using Ring-Closing Metastasis

The macrocyclization using ring-closing metastasis is below in Scheme11.

Example 10 Synthesis of ZG-001

Synthesis of linker ZG-002. The designed linker ZG-002 was prepared byprotection of the amine group of ZG-001 with FMOC-OSu similar toreported synthetic strategy. Briefly, ZG-001 (115 mg, 1 mmol) was in 10%Na₂CO₃ (2.4 mL) and cooled in an ice bath. A solution of Fmoc-OSu (337mg, 1 mmol) in dioxane (3 mL) was then added in 3 portions understirring over a period of 30 min. After further 4 h at room temperature,the reaction mixture was diluted with water (30 mL) and then extractedwith ether (30 mL, 3 times). The remaining aqueous phase was cooled,acidified to pH2.0 with IN HCl, and extracted with ethyl acetate (30 mL,3 times). The organic phase was combined, dried with Na₂SO₄, andevaporated under reduced pressure. The resulting residue wasrecrystallized from EtOAc/hexane to give white crystals (250 mg, 74%yield). ¹H-NMR (DMSO-d6): 2.99(d, J=6.4 Hz, 2H, CH₂COO); 3.60(t, J=5.2Hz, 2H, CH₂N); 4.22(d, J=6.8 Hz, 1H); 4.29(m, 2H, CH₂OCO); 5.55(m, 2H,CH═CH); 7.33-7.88(m, 8H, aryl); 7.52(t, J=5.6 Hz, 1H, NH); 12.24(s, 1H,OH).

Synthesis of linear product ZG-003. The linear product ZG-003 wassynthesized using 9-fluoronylmethoxycarbonyl (Fmoc)-protected aminoacids according to standard SPPS protocols. Briefly, the designed linkerZG-002 was loaded to 2-chlorotrityl resin and followed by coupling withFmoc-Sar-OH, Fmoc-Phe-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-D-homoPhe-OH,and mimic FKBD. After the final coupling, the resin was washed with DCM(2 mL, 3 times), DMF (2 mL, 3 times), MeOH (2 mL, 3 times) and dried for2 h. The dry resin was then suspended in a cleavage cocktail solution ofacetic acid/trifluoroethanol/DCM (1:1:3, 3 mL) and shaken for 4 h tocleave the linear product from the resin. The resulting solution wascollected and concentrated under reduced pressure. Cold etherprecipitation of the residue, followed by HPLC purification gave thefinal product (87% yield). The lyophilized pure product was analyzed byESI-MS. ESI-MS calculated mass for ZG-003 (C₅₃H₆₇N₇O₁₂): 993.5, found[M+Na] 1016.4 m/z.

Synthesis of cyclic product ZG-004. The cyclic product ZG-004 wassynthesized using ring-closing metathesis reaction. In a microwave tube,ZG-003(2 mg, 2 μmol) was dissolved in DCE (0.4 mL) and then a solutionof Hoveyda-Grubbs 2^(nd) generation catalyst (20%) in DCE was added. Thereaction was heated in a microwave synthesizer at 120° C. for 30 min.The reaction mixture was purified by HPLC and the lyophilized pureproduct (46% yield) was analyzed by ESI-MS. ESI-MS calculated mass forZG-004 (C₅₀H₆₃N₇O₁₀): 921.5, found [M+Na] 944.5 m/z.

Example 11 The Design of Rapafucin Library (on Solid Support Beads)—OneBead One Compound (OBOC)

The general structure of rapafucin consist of a) the FKBP-binding domain(FKBD) and b) a peptide chain that cyclizes rapafucin with the FKBD. Thesolid support beads and the linker in connection with the beads andrapafucin are optional for the ease of selection, synthesis and/orpurification. The below figure demonstrates the general structure ofrapafucin library on solid support beads. FKBP-binding domain (FKBD) ishighlighted in red. AA=amino acid

The FKBP-binding domain (FKBD) is obtained either from chemicalsyntheses or degradation reactions with a natural product which belongsto a group comprising tacrolimus (FK-506) and rapamycin. In oneembodiment, FKBD is from degradation reactions of rapamycin.

The peptide moiety is covalently attached to the FKBD. These covalentbonds belong to a group comprising alkene group, amide and thioetherbond. In one embodiment, the covalent bonds are two amide bonds; yet inanother embodiment, a thioether bond and an amide bond. The peptideconsists of 4 or 5 amino acids (AA₁, AA₂, AA₃, . . . , AA_(n); n=4 or 5)which selected arbitrarily from the group comprising but not limited toL-alanine, L-valine, L-phenylalanine, L-glycine, L-methionine,L-proline, L-leucine, L-isoleucine, L-tyrosine, L-threonine,L-asparagine, L-omithine, D-leucine, D-methionine, D-phenylalanine,D-valine, D-homophenylalanine, D-proline, beta-alanine,cyclohexyl-L-alanine, aminoisobutyric acid, 2-aminobenzoic acid,1-aminocyclohexane carboxylic acid, 4-fluoro-L-phenylalanine,4-nitro-L-phenylalanine, L-citrulline, sarcosine, N-methyl-L-leucine,N-methyl-L-norleucine, N-methyl-O-benzyl-L-serine,N-methyl-L-phenylalanine, N-methyl-L-alanine, N-methyl-L-isoleucine,N-methyl-L-valine, N-methyl-O-tert-butyl-L-threonine andN-methyl-O-tert-butyl-L-serine.

The hydroxy group on the cyclohexane moiety of the FKBP-binding domain(FKBD) of rapamycin is designed as a chemical handle linked to the solidsupport beads. The linker belongs to a group comprising silicon-basedlinker, polyethylene glycol (PEG) and aliphatic (C4-C12) esters. In oneembodiment, the linker is a succinate diester. The material of the solidsupport beads belongs to a group polystyrene (PS), TentaGel, HydroGeland controlled pore glass (CPG). In one embodiment, the matrix materialof the solid support beads is polystyrene; yet in another embodiment,TentaGel.

The degradation reactions were carried on a product, rapamycin based onpublished protocol. The carboxyl group on the TBS protected FKBD 2 wascoupled to Fmoc protected ethylenediamine in the presence of a couplingreagent (selected from a group comprising DCC, EDCI, HBTU, HATU) and anon-nucleophilic base. In one embodiment, EDCI and DIPEA were used asthe reagents to synthesize intermediate 3. This reaction was carried at<10° C. for minimizing the side-reactions. In one embodiment, thereaction temperature was 0° C. Upon silica-gel column purification, thematerial was treated with HCl in an organic/aqueous solvent mixture. Inone embodiment, a solvent mixture of THF/H₂O (v/v)=4/1 was chosen. Oncethe selectively deprotected FKBD 4 was obtained and purified by columnchromatography, the material was then co-heated with catalytic amount(10 mol %) of DMAP in pyridine. The optimal condition of the reactionwas stirring at 45° C. for 16 hours. The resulting carboxylic acid 5 wassubsequentially purified by a silica-gel column and a reverse phase HPLCcolumn. H-NMR of compound 5 was measured to verify the purity of thecompound.

Anhydrous intermediate 5 was coupled to a trityl chloride-modified solidsupport in the presence of DIPEA in dichloromethane. In one embodiment,a commercially available polystyrene trityl chloride resin was used; yetin another embodiment, a TentaGel trityl hydroxide resin was used uponbeing activated with thionyl chloride. The loading of compound 5 onsolid support is controlled at 0.10-1.0 mmol/g before quenched by DIPEA(5%) in methanol.

Example 12 Synthesis of FKBD Moiety of on Solid Support Beads

The synthesis of FKBD moiety of rapafucin on support beads is shownbelow in Scheme 12.

With key intermediate 6 on the resin at hand, standard peptide synthesison solid support was conducted for all analogs of rapafucin. Byrepeating steps a (cleavage of N-terminus protecting group) and b(coupling of the N-terminus with a new carboxyl group) (Scheme 2), thepeptide chain was elongated to 4-5 amino acids to yield intermediate 8.Two strategies were explored to cyclize the linear rapafucin 8.

For strategy 1, a sulfur-iodo displacement reaction was applied (Scheme2). Upon coupling of an S-protected 2-mercaptoacetic acid to theN-terminus of intermediate 8 with standard protocol, the aldehyde groupon the tetrahydropyran moiety of FKBD was reduced to a hydroxy group,which is able to be converted to an iodo group later on. The macrocycleis formed in a cascade manner after 9-methylene-9H-fluorene group iscleaved by piperidine (Scheme 2). The rapafucin analog 11 are finalizedby deprotecting TBS group with TBAF at 0° C. Higher temperature orhigher concentration may lead to a nucleophilic cleavage of the esterbond on FKBD. Rapafucins 14-16 were synthesized on strategy 1 with ayield of 4-5%.

For strategy 2, a lactam formation strategy was implemented as shownbelow in Schemes 13-15.

Peptide synthesis/cyclization of rapafucin. Conditions: a) 20%piperidine, DMF, room temperature, 0.2-2 h; b) N-Fmoc protected aminoacid, HATU, DIPEA, DMF, room temperature, 1-3 h; c) t-BuNH₂-BH₃, CH₂Cl₂,room temperature, 20 min; d) Me(PhO)₃PI, 2,6-lutidine, DMF, roomtemperature, 20 min; e) TBAF (0.10 mM), THF, 0° C., 1-2 h; f) oxone,DMF, room temperature, 16 h; g) PyBop, HOAt, DIPEA, DMF, roomtemperature, 3 h. A=Me or H is highlighted in red.

The above-mentioned aldehyde group was herein oxidized into a carboxylgroup before the N-terminus amine was deprotected by standard protocol.The linear amino acid 12 was then treated with macrolactamizationcoupling reagent (e.g., PyBop//DIPEA), and finalized with TBAFdeprotection of the TBS group to give rapafucin analog 13. Rapafucins17-19 were synthesized on strategy 1 with a yield of 4-30% as shownbelow in Scheme 16.

Compound 2 (0.45 g) was prepared by adding EDCI-HCl (128 mg) and DIPEA(333 μL) in DMF (15 mL) at 0° C. for 10 minutes before addingFmoc-ethylenediamine HCl salt (185 mg) in DMF (2 mL). The resultingsolution was stirred at 0° C. for 3 hours and quenched by the additionof HCl (5% aqueous solution, 10 mL). The reaction mixture was dilutedwith 30 mL EtOAc and poured into a separatory funnel. The organic layerwas subsequentially washed with HCl (5% aqueous solution, 30 mL×3) anddried over Na₂SO₄ before being concentrated in vacuo. The crude productwas purified by silica-gel column chromatography eluting with MeOH inCH₂Cl₂ (2-5%). 294 mg product (68%) was obtained as a yellow solid.

Although the invention has been described with reference to the aboveexample, it may be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

What is claimed is:
 1. A method of identifying compounds from a libraryof compounds that bind to a target protein optionally in complex withFKBP, the method comprising: a) synthesizing a hybrid combinatoriallibrary of compounds containing an FKBP binding domain and peptide ornon-peptide effector domain bound to resin in a one-bead-one-compoundlibrary; b) screening the one-bead-one-compound library by incubatingthe one-bead-one-compound library with the target protein optionally inthe presence of FKBP; wherein the target protein is optionally labeledwith the fluorescent label; c) isolating the specific beads from theone-bead-one-compound library complexed with the fluorescently labeledprotein; and d) determining the identity of the compounds on theisolated beads from the one-bead-one-compound library.
 2. The method ofclaim 1, wherein the library of compounds has Formula I:

or a pharmaceutically acceptable salt or solvate thereof, wherein:

is a single or double bond; X₁ is O or NR⁶; Y is —C(O)— or

X₂ is (CH₂)_(m), O, or NR⁶; Z is

W is O, CH, CH₂, CR⁴, or CR⁵; L₁ and L₂ are each independently a directbond, substituted or unsubstituted —(C₁-C₆)alkyl-, substituted orunsubstituted —(CH₂)_(n)O(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)C(O)—, substituted or unsubstituted—(CH₂)_(n)C(O)(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)C(O)O(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)NH(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)S(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₁-C₆)alkyl-, substituted or unsubstituted—(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)O(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)C(O)O(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)NH(C₁-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)S(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkenyl-, substituted or unsubstituted—(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)O(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)C(O)O(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)NH(C₁-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)S(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkynyl-, wherein each alkyl, alkenyl andalkynyl group may be optionally substituted with alkyl, alkoxy, amino,carboxyl, cyano, nitro, or trifluoromethyl; each m is independently aninteger selected from 0, 1, 2, 3, 4, 5, and 6; each n is independentlyan integer selected from 0, 1, 2, 3, 4, 5, and 6; R¹ is

wherein

is a resin; R² is hydrogen, hydroxyl, or alkoxy; R³ is hydrogen oralkyl; R⁴ and R⁵ are each independently hydrogen, hydroxy, alkyl,alkoxy, or OPG, wherein PG is a protecting group; R⁶ is hydrogen oralkyl; wherein the Effector Domain has Formula II:

wherein: R⁷, R⁹, R¹¹, R¹³ and R¹⁵ are each independently hydrogen oralkyl; R⁸, R¹⁰, R¹², and R¹⁴ are each independently hydrogen, halogen,amino, cyano, nitro, trifluoromethyl, substituted or unsubstitutedalkyl, substituted or unsubstituted alkenyl, substituted orunsubstituted alkynyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted perfluoroalkyl, substituted orunsubstituted alkoxy, substituted or unsubstituted alkylamino,substituted or unsubstituted alkylthio, substituted or unsubstitutedaryl, substituted or unsubstituted alkylaryl, substituted orunsubstituted heteroalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted heteroaryl, substitutedor unsubstituted heteroalkylaryl, (CH₂)_(n)CN, (CH₂)_(n)CF₃,(CH₂)_(n)C₂F₅, (CH₂)_(n)OR¹⁶, (CH₂)_(n)C(O)R¹⁶, (CH₂)_(n)C(O)OR¹⁶,(CH₂)_(n)OC(O)R¹⁶, (CH₂)_(n)NR¹⁷R¹⁸, (CH₂)_(n)C(O)NR¹⁷R¹⁸,(CH₂)_(n)N¹⁹RC(O)R¹⁶, (CH₂)_(n)N¹⁹RC(O)OR¹⁶, (CH₂)_(n)NR¹⁹C(O)NR¹⁷R¹⁸,(CH₂ _(n)SR¹⁶, (CH₂)_(n)S(O)_(j)NR¹⁷R¹⁸, (CH₂)_(n)N¹⁹RS(O)_(j)R¹⁶, or—(CH₂)_(n)NR¹⁹S(O)_(j)NR¹⁷R¹⁸; n is an integer selected from 0, 1, 2, 3,4, 5, and 6; j is an integer selected from 0, 1, and 2; R¹⁶, R¹⁷, R¹⁸,and R¹⁹ are each independently hydrogen, halogen, amino, cyano, nitro,trifluoromethyl, alkyl, alkenyl, alkynyl, cycloalkyl, perfluoroalkyl,alkoxy, alkylamino, alkylthio, aryl, alkylaryl, heteroalkyl,heterocycloalkyl, heteroaryl, or heteroalkylaryl, or R¹⁶ and R¹⁹ are asdescribed above, and R¹⁷ and R¹⁸, together with the N atom to which theyare attached, form a substituted or unsubstituted 5-, 6-, or 7-memberedheterocycloalkyl or a substituted or unsubstituted 5-memberedheteroaryl, wherein each of the above groups listed for R⁸, R¹⁰, R¹²,and R¹⁴ may be optionally independently substituted with 1 to 3 groupsselected from halogen, amino, cyano, nitro, trifluoromethyl, alkyl,alkenyl, alkynyl, cycloalkyl, perfluoroalkyl, alkoxy, alkylamino,alkylthio, aryl, alkylaryl, heteroalkyl, heterocycloalkyl, heteroaryl,heteroalkylaryl, (CH₂)_(n)CN, (CH₂)_(n)CF₃, (CH₂)_(n)C₂F₅,(CH₂)_(n)OR¹⁶, (CH₂)_(n)C(O)R¹⁶, (CH₂)_(n)C(O)OR¹⁶, (CH₂)_(n)OC(O)R¹⁶,(CH₂)_(n)NR¹⁷R¹⁸, (CH₂)_(n)C(O)NR¹⁷R¹⁸, (CH₂)_(n)N¹⁹RC(O)R¹⁶,(CH₂)_(n)N¹⁹RC(O)OR¹⁶, (CH₂)_(n)NR¹⁹C(O)NR¹⁷R¹⁸, (CH₂)_(n)SR¹⁶,(CH₂)_(n)S(O)_(j)NR¹⁷R¹⁸, (CH₂)_(n)N¹⁹RS(O)_(j)R¹⁶, and—(CH₂)_(n)NR¹⁹S(O)_(j)NR¹⁷R¹⁸.
 3. The method of claim 1, wherein in thecompound of Formula I: X is O or NR⁶; L₁ and L₂ are each independently—(C₁-C₆)alkyl-, —(CH₂)_(n)O(C₁-C₆)alkyl-, —(CH₂)_(n)C(O)(C₁-C₆)alkyl-,—(CH₂)_(n)C(O)O(C₁-C₆)alkyl-, —(CH₂)_(n)NH(C₁-C₆)alkyl-,—(CH₂)_(n)C(O)NH(C₁-C₆)alkyl-, —(CH₂)_(n)S(C₁-C₆)alkyl-,—(CH₂)_(n)C(O)(CH₂)_(n)S(C₁-C₆)alkyl-, —(C₂-C₆)alkenyl-,—(CH₂)_(n)O(C₂-C₆)alkenyl-, —(CH₂)_(n)C(O)(C₂-C₆)alkenyl-,—(CH₂)_(n)C(O)O(C₂-C₆)alkenyl-, —(CH₂)_(n)NH(C₁-C₆)alkenyl-,—(CH₂)_(n)C(O)NH(C₂-C₆)alkenyl-, —(CH₂)_(n)S(C₂-C₆)alkenyl-,—(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkenyl-, wherein each alkyl and alkenylgroup may be substituted with alkyl, alkoxy, or carboxyl; n is aninteger selected from 0, 1, 2, 3, 4, 5, and 6; R¹ is

wherein

is a resin; R² is hydroxyl or alkoxy; R³ is hydrogen or alkyl; R⁴ and R⁵are each independently hydrogen, alkyl, alkoxy, or OPG, wherein PG is asilyl protecting group; R⁶ is hydrogen; R⁷, R⁹, R¹¹, R¹³, and R¹⁵ areeach independently hydrogen or CH₃; R⁸, R¹⁰, R¹², and R¹⁴ are eachindependently substituted or unsubstituted cyclopropyl, substituted orunsubstituted cyclobutyl, substituted or unsubstituted cyclopentyl,substituted or unsubstituted cyclohexyl, substituted or unsubstitutedcycloheptyl, or substituted or unsubstituted cyclooctyl; or R⁸, R¹⁰,R¹², and R¹⁴ are each independently substituted or unsubstitutedtetrahydrofuranyl, substituted or unsubstituted tetrahydrothiophenyl,substituted or unsubstituted pyrrolidinyl, substituted or unsubstituted1,3-dioxolanyl, substituted or unsubstituted pyrazolidinyl, substitutedor unsubstituted imidazolidinyl, substituted or unsubstituted1,4-dioxanyl, substituted or unsubstituted piperidinyl, substituted orunsubstituted piperazinyl, substituted or unsubstituted morpholinyl,substituted or unsubstituted thiomorpholinyl, or substituted orunsubstituted 1,4-dithianyl; or R⁸, R¹⁰, R¹², and R¹⁴ are eachindependently substituted or unsubstituted phenyl, substituted orunsubstituted benzyl, substituted or unsubstituted naphthylenyl, orsubstituted or unsubstituted biphenyl; or R⁸, R¹⁰, R¹², and R¹⁴ are eachindependently substituted or unsubstituted furanyl, substituted orunsubstituted thiophenyl, substituted or unsubstituted pyrrolyl,substituted or unsubstituted pyrazolyl, substituted or unsubstitutedimidazolyl, substituted or unsubstituted triazolyl, substituted orunsubstituted isoxazolyl, substituted or unsubstituted oxazolyl,substituted or unsubstituted thiazolyl, substituted or unsubstitutedisothiazolyl, substituted or unsubstituted pyridinyl, substituted orunsubstituted pyridizanyl, substituted or unsubstituted pyrimidinyl,substituted or unsubstituted triazinyl, substituted or unsubstitutedbenzofuranyl, substituted or unsubstituted benzo(b)thiophenyl,substituted or unsubstituted indolyl, substituted or unsubstitutedbenzimidazolyl, substituted or unsubstituted indazolyl, substituted orunsubstituted benzisoxazolyl, substituted or unsubstituted benzoxazolyl,substituted or unsubstituted benzothiazolyl, substituted orunsubstituted quinolinyl, substituted or unsubstituted isoquinolinyl,substituted or unsubstituted quinazolinyl, substituted or unsubstitutedquinoxalinyl, or substituted or unsubstituted naphthyridinyl.
 4. Themethod of claim 1, wherein in the compound of Formula I: L₁ and L₂ areeach independently —(C₁-C₆)alkyl-, —(CH₂)_(n)O(C₁-C₆)alkyl-,—(CH₂)_(n)C(O)(C₁-C₆)alkyl-, —(CH₂)_(n)NH(C₁-C₆)alkyl-,—(CH₂)_(n)C(O)NH(C₁-C₆)alkyl-, —(C₂-C₆)alkenyl-,—(CH₂)_(n)O(C₂-C₆)alkenyl-, —(CH₂)_(n)C(O)(C₂-C₆)alkenyl-,—(CH₂)_(n)NH(C₁-C₆)alkenyl-, —(CH₂)_(n)C(O)NH(C₂-C₆)alkenyl-, whereineach alkyl and alkenyl group may be substituted with alkyl, alkoxy, orcarboxyl; n is an integer selected from 0, 1, 2, 3, 4, 5, and 6; R¹ is

wherein

is Wang resin; R⁴ and R⁵ are each independently hydrogen, hydroxy,alkyl, alkoxy, or OPG, wherein PG is a tert-butyldimethylsilylprotecting group; R⁷, R⁹, R¹¹, R¹³, and R¹⁵ are each independentlyhydrogen; R⁸, R¹⁰, R¹², and R¹⁴ are each independently hydrogen,substituted or unsubstituted alkyl, substituted or unsubstituted benzyl,substituted or unsubstituted pyrrolidinyl, substituted or unsubstitutedindolyl, (CH₂)_(n)OR⁵, (CH₂)_(n)C(O)NR⁶R⁷, or (CH₂)_(n)SR⁵; and R¹⁶,R¹⁷, and R¹⁸ are each independently hydrogen or (C₁-C₆)alkyl.
 5. Themethod of claim 1, wherein in the compound of Formula I: L₁ and L₂ areeach independently —(C₁-C₆)alkyl-, —O(C₁-C₆)alkyl-, —C(O)(C₁-C₆)alkyl-,—(CH₂)_(n)C(O)NH(C₁-C₆)alkyl-, —(C₂-C₆)alkenyl-, —O(C₂-C₆)alkenyl-,—C(O)(C₂-C₆)alkenyl-, —(CH₂)_(n)C(O)NH(C₂-C₆)alkenyl-, wherein eachalkyl and alkenyl group may be substituted with alkyl, alkoxy, orcarboxyl; n is an integer selected from 0, 1, 2, 3, 4, 5, and 6; R⁸,R¹⁰, R¹², and R¹⁴ are each independently H, CH₃, CH₂OH, CH₂SH,CH(OH)CH₃, CH₂C(O)NH₂, CH₂CH₂C(O)NH₂, CH₂CH₂SCH₃, CH₂CH(CH₃)₂,CH(CH₃)CH₂CH₃, CH₂C₆C₅,


6. The method of claim 1, wherein in the compound of Formula I: L₁ andL₂ are each independently —OCH₂CH₂—, —CH₂C(O)—, —CH₂CH₂C(O)—,—C(O)NHCH₂CH₂, —CH₂CH═CHCH₂—, —OCH₂CH═CHCH₂CH₂—, —OCH₂CH═CHCH₂CH(CO2H)—,—CH₂C(O)NHCH₂CH₂—, or CH₂CH(OCH₃)═C(CH₃)CH₂CH₂; and R⁸, R¹⁰, R¹², andR¹⁴ are each independently the sidechain of the amino acid alanine,asparagine, cysteine, glutamine, glycine, isoleucine, leucine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, or valine.
 7. A method of validating the compounds identifiedin the method of claim 1, the method comprising: a) synthesizing acompound of Formula I:

or a pharmaceutically acceptable salt or solvate thereof, wherein:

is a single or double bond; X₁ is O or NR⁶; Y is —C(O)— or

X₂ is (CH₂)_(m), O, or NR⁶; Z is

W is O, CH, CH₂, CR⁴, or CR⁵; L₁ and L₂ are each independently a directbond, substituted or unsubstituted —(C₁-C₆)alkyl-, substituted orunsubstituted —(CH₂)_(n)O(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)C(O)—, substituted or unsubstituted—(CH₂)_(n)C(O)(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)C(O)O(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)NH(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)S(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₁-C₆)alkyl-, substituted or unsubstituted—(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)O(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)C(O)O(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)NH(C₁-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)S(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkenyl-, substituted or unsubstituted—(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)O(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)C(O)O(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)NH(C₁-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)S(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkynyl-, wherein each alkyl, alkenyl andalkynyl group may be optionally substituted with alkyl, alkoxy, amino,carboxyl, cyano, nitro, or trifluoromethyl; each m is independently aninteger selected from 0, 1, 2, 3, 4, 5, and 6; each n is independentlyan integer selected from 0, 1, 2, 3, 4, 5, and 6; R¹ is hydrogen,hydroxyl, or OPG, wherein PG is a protecting group; R² is hydrogen,hydroxyl, or alkoxy; R³ is hydrogen or alkyl; R⁴ and R⁵ are eachindependently hydrogen, hydroxy, alkyl, alkoxy, or OPG, wherein PG is aprotecting group; R⁶ is hydrogen or alkyl; wherein the Effector Domainhas Formula II:

wherein: R⁷, R⁹, R¹¹, R¹³, and R¹⁵ are each independently hydrogen oralkyl; R⁸, R¹⁰, R¹², and R¹⁴ are each independently hydrogen, halogen,amino, cyano, nitro, trifluoromethyl, substituted or unsubstitutedalkyl, substituted or unsubstituted alkenyl, substituted orunsubstituted alkynyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted perfluoroalkyl, substituted orunsubstituted alkoxy, substituted or unsubstituted alkylamino,substituted or unsubstituted alkylthio, substituted or unsubstitutedaryl, substituted or unsubstituted alkylaryl, substituted orunsubstituted heteroalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted heteroaryl, substitutedor unsubstituted heteroalkylaryl, (CH₂)_(n)CN, (CH₂)_(n)CF₃,(CH₂)_(n)C₂F₅, (CH₂)_(n)OR¹⁶, (CH₂)_(n)C(O)R¹⁶, (CH₂)_(n)C(O)OR¹⁶,(CH₂)_(n)OC(O)R¹⁶, (CH₂)_(n)NR¹⁷R¹⁸, (CH₂)_(n)C(O)NR¹⁷R¹⁸,(CH₂)_(n)N¹⁹RC(O)R¹⁶, (CH₂)_(n)N¹⁹RC(O)OR¹⁶, (CH₂)_(n)NR¹⁹C(O)NR¹⁷R¹⁸,(CH₂)_(n)SR¹⁶, (CH₂)_(n)S(O)_(j)NR¹⁷R¹⁸, (CH₂)_(n)N¹⁹RS(O)_(j)R¹⁶, or—(CH₂)_(n)NR¹⁹S(O)_(j)NR¹⁷R¹⁸; n is an integer selected from 0, 1, 2, 3,4, 5, and 6; j is an integer selected from 0, 1, and 2; R¹⁶, R¹⁷, R¹⁸,and R¹⁹ are each independently hydrogen, halogen, amino, cyano, nitro,trifluoromethyl, alkyl, alkenyl, alkynyl, cycloalkyl, perfluoroalkyl,alkoxy, alkylamino, alkylthio, aryl, alkylaryl, heteroalkyl,heterocycloalkyl, heteroaryl, or heteroalkylaryl, or R¹⁶ and R¹⁹ are asdescribed above, and R¹⁷ and R¹⁸, together with the N atom to which theyare attached, form a substituted or unsubstituted 5-, 6-, or 7-memberedheterocycloalkyl or a substituted or unsubstituted 5-memberedheteroaryl, wherein each of the above groups listed for R⁸, R¹⁰, R¹²,and R¹⁴ may be optionally independently substituted with 1 to 3 groupsselected from halogen, amino, cyano, nitro, trifluoromethyl, alkyl,alkenyl, alkynyl, cycloalkyl, perfluoroalkyl, alkoxy, alkylamino,alkylthio, aryl, alkylaryl, heteroalkyl, heterocycloalkyl, heteroaryl,heteroalkylaryl, (CH₂)_(n)CN, (CH₂)_(n)CF₃, (CH₂)_(n)C₂F₅,(CH₂)_(n)OR¹⁶, (CH₂)_(n)C(O)R¹⁶, (CH₂)_(n)C(O)OR¹⁶, (CH₂)_(n)OC(O)R¹⁶,(CH₂)_(n)NR¹⁷R¹⁸, (CH₂)_(n)C(O)NR¹⁷R¹⁸, (CH₂)_(n)N¹⁹RC(O)R¹⁶,(CH₂)_(n)N¹⁹RC(O)OR¹⁶, (CH₂)_(n)NR¹⁹C(O)NR¹⁷R¹⁸, (CH₂)_(n)SR¹⁶,(CH₂)_(n)S(O)_(j)NR¹⁷R¹⁸, (CH₂)_(n)N¹⁹RS(O)_(j)R¹⁶, and—(CH₂)_(n)NR¹⁹S(O)_(j)NR¹⁷R¹⁸.
 8. The method of claim 1, wherein theidentity of the compounds on the isolated beads from theone-bead-one-compound library is determined using mass spectrometry.